Method for the diagnosis of aneurysm, and inhibitors for use in the prevention or treatment of aneurysm

Abstract

The present invention refers to an in vitro method for the diagnosis of aneurysm which comprises assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl-glucosamine-1-P or UDP-N-acetyl- glucosamine, Galactosamine-6-P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-1-P or UDP-N-acetyl-galactosamine or of proteins that have been co-translationally or post- translationally modified trough the addition of the above nucleotide sugars. It also refers to an inhibitor of an enzyme involved in the hexosamine biosynthetic pathway selected from the group comprising: GFPT1, GFPT2, GNPNAT1, PGM3 and / or UAP1, and / or of an enzyme downstream the hexosamine pathway selected from: GALE, OGT, OGA, MGAT1, MGAT2, MGAT3, MGAT5B, B3GNT3, GNE, HAS1, HAS3 and / or enzymes involved in the integrated stress response (ISR), for use in the prevention and / or treatment of aneurysm.

Description

[0001] METHOD FOR THE DIAGNOSIS OF ANEURYSM, AND INHIBITORS FOR USE IN THE PREVENTION OR TREATMENT OF ANEURYSM

[0002] FIELD OF THE INVENTION

[0003] The present invention refers to the medical field. Particularly, the present invention refers to an in vitro method for the diagnosis of aneurysm which comprises assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl-glucosamine-l-P or UDP-N-acetyl- glucosamine, Galactosamine-6-P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine or of proteins that have been co-translationally or post- translationally modified trough the addition of the above nucleotide sugars. It also refers to an inhibitor of an enzyme involved in the hexosamine biosynthetic pathway selected from the group comprising: GFPT1, GFPT2, GNPNAT1, PGM3 and / or UAP1, and / or of an enzyme downstream the hexosamine pathway selected from: GALE, OGT, OGA, MGAT1, MGAT2, MGAT3, MGAT5B, B3GNT3, GNE, HAS1, HAS3 and / or enzymes involved in the integrated stress response (ISR), for use in the prevention and / or treatment of aneurysm.

[0004] STATE OF THE ART

[0005] The aorta is the body’s largest artery. It carries oxygen-rich blood from the heart to the rest of the body. This blood vessel originates in the heart’s left ventricle, or upper chamber, and then curves upward into the chest before bending downward into the abdomen, where it splits into two arteries that carry blood to each leg. Other arteries branch off from parts of the aorta to carry blood to the upper body and to organs such as the kidneys, stomach, and intestines.

[0006] The wall of the aorta is composed of three layers. If the middle layer of the aortic wall - which consists of smooth muscle and elastic tissue - weakens and stretches, blood pumping through it can lead to a bulge, or aneurysm.

[0007] Small aneurysms generally do not cause problems, but larger ones can cause blood to clot or an artery to rupture or tear, known as dissection. This is a life-threatening event that requires immediate surgery. There are three types of aneurysms based on their location on the aorta: thoracic, abdominal, and thoracoabdominal.

[0008] Aortic aneurysms can be triggered by genetic disorders such as Marfan syndrome (MFS) and related aortic diseases as well as by inflammatory disorders such as giant cell arteritis or atherosclerosis. In all these conditions, cardiovascular risk factors, such as systemic arterial hypertension, may contribute to faster rate of aneurysm progression.

[0009] The diagnosis of aortic aneurism is generally made with ultrasound, although a CT scan or an MRI is needed to provide more details about the shape or location of the aneurysm. However, aortic aneurysms can be very difficult to detect through physical examination and may go undetected for years unless specifically tested. In many cases aortic aneurysm has no symptoms, and it is not diagnosed until there is a life-threatening medical emergency.

[0010] So, there is an unmet medical need of developing strategies for the diagnosis and treatment of aneurysm. The present invention is focused on solving this problem, and both a method to diagnose aneurysm and a therapeutic strategy are herein provided.

[0011] DESCRIPTION OF THE INVENTION

[0012] Brief description of the invention

[0013] As explained above, the present invention refers to an in vitro method for the diagnosis of aneurysm which comprises assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N-acetyl- glucosamine-6P, N-acetyl-glucosamine-l-P or UDP-N-acetyl-glucosamine, Galactosamine-6- P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine or of proteins that have been co-translationally or post-translationally modified trough the addition of the above nucleotide sugars. It also refers to an inhibitor of an enzyme involved in the hexosamine biosynthetic pathway selected from the group comprising: GFPT1, GFPT2, GNPNAT1, PGM3 and / or UAP1, and / or of an enzyme downstream the hexosamine pathway selected from: GALE, OGT, OGA, MGAT1, MGAT2, MGAT3, MGAT5B, B3GNT3, GNE, HAS1, HAS3 and / or enzymes involved in the integrated stress response (ISR), for use in the prevention and / or treatment of aneurysm. To study the pathophysiology of aneurism, the inventors of the present invention have performed experiments with Fbnlcl039G / +mice, a mouse model of MFS that rapidly develops thoracic aortic aneurysm and dissection (TAAD).

[0014] The inventors performed a transcriptomic analysis of the aortas of Fbnlcl039G / +mice (Fig. 1A- C). This analysis revealed that, compared to aortic tissue from Fbnl+ / +mice, aortic tissue from Fbnlcl039G / +mice has: an increased expression of enzymes involved in the hexosamine biosynthetic pathway (HBP). In particular, the overexpressed enzymes are: Gfpll, Gfpt2, Uapl and Gnpatl. an increased expression of enzymes involved in O-linked-N-acetylglucosaminylation (O-GlcNAcylation), which is a type of glycosylation that occurs when the monosaccharide O-GlcNAc is attached to the oxygen atom of serine or threonine residues of nuclear or cytoplasmic proteins. In particular, the enzymes are: Ogt and O a. an increased expression of enzymes involved in N-linked glycosylation in aortas of Fbn lc 1039G / +mice. N-liked glycosylation occurs when an oligosaccharide is added onto a nitrogen atom of an asparagine residue of a protein. N-glycosylation often occurs co- translationally, in that the glycan is attached to the nascent protein as it is being translated and transported into the endoplasmic reticulum (ER). In particular, the enzymes are: Mgatl, Mgat2, Mgat3, Mgat5b, B3gnt3, Gne, Hasl andHas3.

[0015] Of note, the above-mentioned pathways / enzymes are closely related, since the HBP pathway results in the synthesis of the sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), and this is an essential common donor substrate for O-GlcNAcylation as well as GlcNAc- branched N-glycosylation.

[0016] In line with the above results: an increased protein expression of GFPT2 and UAP1, as well as an increased content of O-GlcNAc and WGA (a lectin that specifically binds to GlcNAc residues) was detected in aortic tissue from Fbnlcl039G / +mice compared to controls (Fig. 1D-F).

[0017] - Fbnl silencing in primary murine vascular smooth muscle cells (VSMCs) resulted in an increased protein expression of GFPT2 and UAP1, as well as a higher O-GlcNAc content (Fig. II). an increased N-acetylhexosamine and O-GlcNAc content was detected in plasma from FbnlCI039G / +mice compared to controls (Fig.l G, H), indicating that bn i deficiency resulted in an increased abundance of downstream HBP metabolites in plasma.

[0018] These data suggest that TAAD results in an increased expression of the HBP, and downstream enzymes involved in O-GlcNAcylation and N-liked glycosylation in aortic tissue, and in an increased abundance of proteins that have been co-translationally or post-transcriptionally modified trough the addition of the nucleotide sugars produced by the HBP.

[0019] The inventors found that pharmacological induction of HBP increased aortic diameter, decreased blood pressure (BP) (Fig. 2A-D), and damaged aortic architecture in Fbnlcl039G / +and control mice (Fig. 2E), indicating that excessive HBP activity is detrimental for aortic homeostasis.

[0020] The results provided in the present application suggest that excessive HBP activation leads to the induction of the integrated stress response (ISR) (Example 2.4). The inventor found that: aortic tissue from Fbnlcl039G / +mice has an increased expression of genes involved in the ISR compared to their control counterparts (Fig. 4A, F-I), including an increased protein level of p-PERK, p-eIF2a and ATF4 (Fig. 4F, G).

[0021] Fbnl inhibition induces the IRS through p-eIF2a and ATF4 in VSMCs (Fig. 4B, C). GFPT2 overexpression induces the IRS through p-eIF2a and ATF4 in VSMCs (Fig. 4D, E)

[0022] The inventors then investigated the therapeutic potential of HBP inhibition both in vitro and in vivo. They found that treatment with DON (6-diazo-5-oxo-L-norleucine), an HBS inhibitor that blocks GFPT enzymatic activity: reduced O-GlcNAc content in shF7w / -VSMCs (Fig. 3A, B), and the expression of classical MFS genes such as Tgbl, gf2. Sppl and Collal (Fig. 3C). normalized aortic diameter and blood pressure (Fig. 3E, F), restored histologic features of aortic degeneration (Fig. 3G), and reduced O-GlcNAc and WGA content in aortic tissue (Fig. 3H, I) in Fbnlcl039G / +mice. normalized the expression of genes involved in ISR (Fig. 4B, C, F-J). These data indicate that HBP-inhibition improves aortic architecture and homeostasis in a TA AD mouse model.

[0023] The inventors also investigated the therapeutic potential of inhibiting the ISR both in vitro and in vivo. They found that treatment with the ISR-inhibitor ISRIB, which is thought to inhibit the ISR through PERK and EIF2a: reduced Atf4, Atf6 mRNA levels in shFbnJ-VSMCs (Fig. 5A). normalized aortic diameter and systolic blood pressure in Fbnlcl039G / +mice (Fig. 5C).

[0024] These data indicate that ISR inhibition improves aortic architecture and homeostasis in a TAAD mouse model.

[0025] Importantly, the inventors found that aortic sections from MFS-patients also displayed increased levels of GFPT2, O-GlcNAc, WGA and p-PERK (Fig. 6), suggesting that the findings in the above-mentioned in vivo and in vitro models are extensive to human TAAD.

[0026] Finally, the inventors assessed the involvement of the HBS and IRS pathways in a mouse model of acute atherosclerotic abdominal aortic aortic aneurysms (AAA) and ruptures. For this, ApoE- deficient mice were challenged with angiotensin-II infusion for 28 days at three weeks after the initiation of a high cholesterol and fat diet (ApoE^ AAA mice, Fig. 7A). In challenged mice, hypertension and hypercholesterolemia promote robust aneurysm development and lethal aortic rupture. ApoE AAA mice displayed increased mRNA expression of HBS markers such as GFPT2 and UAP1, and the IRS master regulator ATF4 compared with ApoE controlmice (Fig. 7B).

[0027] These data indicate that HBS and ISR pathways are also involved in AAA pathology.

[0028] So, the first embodiment of the present invention refers to an in vitro method for the diagnosis of aneurysm, which comprises: a) assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N- acetyl-glucosamine-l-P or UDP-N-acetyl-glucosamine , Galactosamine-6-P, N-acetyl- galactosamine-6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, or of proteins that have been co-translationally or post-translationally modified trough the addition of a nucleotide sugar selected from the list comprising: Glucosamine-6-P, N- acetyl-glucosamine-6P, N-acetyl-glucosamine-l-P or UDP-N-acetyl-glucosamine, Galactosamine-6-P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-l-P or UDP- N-acetyl -galactosamine, in a biological sample obtained from a subject, b) wherein a higher level, as compared with a pre-established threshold value determined in subjects who are not suffering from aneurysm, is an indication that the subject is suffering from aneurysm.

[0029] In a preferred embodiment the biological sample is selected from: aortic tissue, serum, plasma or blood.

[0030] In a preferred embodiment the proteins that have been co-translationally or post-translationally modified trough the addition of nucleotide sugars are N-linked glycosylated proteins or O- linked glycosylated proteins, preferably O-linked N-acetylglucosamine acylated (O- GlcNAcylated) proteins or proteins comprising N-acetylhexosamines.

[0031] The second embodiment of the invention refers to the in vitro use of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N- acetyl-glucosamine-6P, N-acetyl-glucosamine-l-P or UDP-N-acetyl-glucosamine, Galactosamine-6-P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-l-P or UDP-N- acetyl-galactosamine or of proteins that have been co-translationally or post-translationally modified trough the addition of a nucleotide sugar selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl-glucosamine-l-P or UDP-N-acetyl- glucosamine, Galactosamine-6-P, N-acetyl-galactosamine-6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, or of a kit comprising reagents for the detection of a nucleotide sugar selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl- glucosamine-l-P or UDP-N-acetyl-glucosamine, Galactosamine-6-P, N-acetyl-galactosamine- 6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, or of proteins that have been co-translationally or post-translationally modified trough the addition of a nucleotide sugar selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl- glucosamine-l-P or UDP-N-acetyl-glucosamine, Galactosamine-6-P, N-acetyl-galactosamine- 6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, for the diagnosis of aneurysm.

[0032] The third embodiment of the invention refers to an in vitro method for screening or identifying compounds for use in the prevention and / or treatment of aneurysm, which comprises: a) assessing the enzyme activity of an enzyme involved in the hexosamine biosynthetic pathway selected from: GFPT1, GFPT2, GNPNAT1, PGM3 and / or UAP1, and / or of an enzyme downstream the hexosamine pathway selected from: GALE, OGT, OGA, MGAT1, MGAT2, MGAT3, MGAT5B, B3GNT3, GNE, HAS1, HAS3 and / or an enzyme involved in the integrated stress response (ISR), once the candidate compound has been incubated with the enzyme, and b) wherein if an inhibition of the enzyme is observed, it is indicative that the candidate compound may be effective in the prevention and / or treatment of aneurysm.

[0033] The fourth embodiment of the invention refers to an inhibitor of an enzyme (hereinafter referred to as the inhibitor of the invention) involved in the hexosamine biosynthetic pathway selected from the group comprising: GFPT1, GFPT2, GNPNAT1, PGM3 and / or UAP1, and / or of an enzyme downstream the hexosamine pathway selected from: GALE, OGT, OGA, MGAT1, MGAT2, MGAT3, MGAT5B, B3GNT3, GNE, HAS1, HAS3 and / or an enzyme involved in the ISR, for use in the prevention and / or treatment of aneurysm.

[0034] In a preferred embodiment the enzyme involved in the ISR is selected from the list comprising: PERK and / or EIF2a.

[0035] In a preferred embodiment the inhibitor of an enzyme involved in the hexosamine biosynthetic pathway is selected from: azaserine or DON, and / or wherein the inhibitor of the enzyme downstream the hexosamine pathway is selected from: OSML1 or ISRIB.

[0036] The fifth embodiment of the invention refers to a composition comprising the inhibitor of the invention, optionally, pharmaceutically acceptable excipients or carriers for use in the prevention and / or treatment of aneurysm.

[0037] In a preferred embodiment the aneurysm is selected from aortic aneurysm, abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated to atherosclerosis, or thoracic aortic aneurysm, preferably aneurysm associated to Marfan syndrome.

[0038] The present invention also refers to:

[0039] In vitro method for identifying biomarker signatures for the diagnosis of aneurysm, which comprises assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl- glucosamine-l-P or UDP-N-acetyl -glucosamine, Galactosamine-6-P, N-acetyl -galactosamine- 6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, or of proteins that have been co-translationally or post-translationally modified trough the addition of a nucleotide sugar selected from the list comprising: Glucosamine-6-P, N-acetyl-glucosamine-6P, N-acetyl- glucosamine-l-P or UDP-N-acetyl -glucosamine, Galactosamine-6-P, N-acetyl-galactosamine- 6P, N-acetyl-galactosamine-l-P or UDP-N-acetyl-galactosamine, in a biological sample obtained from a subject, wherein a higher level, as compared with a pre-established threshold value determined in subjects who are not suffering from aneurysm, is an indication that the it is a reliable biomarker.

[0040] In a preferred embodiment, the method is focused on detecting biomarkers in a test sample from a human subject at risk of aneurysm.

[0041] In a preferred embodiment, the method is complemented by using image techniques like, for example, CT scan or an MRI. Particularly, once an aortic aneurysm is seen or suspected on ultrasound, CT scan or an MRI can be obtained to provide more details about the shape or location of the aneurysm.

[0042] In a preferred embodiment, the present invention is a computer-implemented invention, wherein a processing unit (hardware) and a software are configured to: Receive values of any of the above cited biomarkers or signatures, process the values received for finding substantial variations or deviations, and provide an output through a terminal display of the variation or deviation of the concentration level.

[0043] Particularly, the present invention refers to an in vitro method for the diagnosis of aortic aneurysm, which comprises: a) Assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising or consisting of: N-AcetylGlucosamine, N-AcetylGalactosamine or glucosamine, or glycosaminoglycans comprising thereof, in a biological sample obtained from a subject, b) wherein a higher level, as compared with a pre- established threshold value determined in subjects who are not suffering from aortic aneurysm, is an indication that the subject is suffering from aortic aneurysm.

[0044] The present invention also refers to the in vitro use of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list comprising: N-AcetylGlucosamine, N- AcetylGalactosamine and glucosamine or glycosaminoglycans comprising thereof, or of a kit comprising reagents for the detection of a nucleotide sugar selected from the list comprising: N-AcetylGlucosamine, N-AcetylGalactosamine and glucosamine, or glycosaminoglycans comprising thereof, for the diagnosis of aortic aneurysm.

[0045] The present invention also refers to an inhibitor of an enzyme involved in the hexosamine biosynthetic pathway and / or of an enzyme downstream the hexosamine pathway and / or an enzyme involved in the integrated stress response (ISR), selected from FR054 with CAS No. : 35954-65-5, 6-diazo-5-oxo-norleucine (DON) or ISRIB with CAS No.: 1597403-47-8, or a pharmaceutical composition comprising thereof, for use in a method for the prevention and / or treatment of aortic aneurysm. Alternatively, the present invention refers to a method for the treatment of aortic aneurysm which comprises the administration of a therapeutically effective dose of an inhibitor of an enzyme involved in the hexosamine biosynthetic pathway and / or of an enzyme downstream the hexosamine pathway and / or an enzyme involved in the integrated stress response (ISR), selected from FR054 with CAS No.: 35954-65-5, 6-diazo-5-oxo- norleucine (DON) with CAS No.: 157-03-9 or ISRIB with CAS No.: 1597403-47-8.

[0046] In a preferred embodiment, the aortic aneurysm is selected from abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated to atherosclerosis, or thoracic aortic aneurysm, preferably aneurysm associated to Marfan syndrome.

[0047] In a preferred embodiment, the biological sample is selected from: aortic tissue, serum, plasma or blood.

[0048] In the context of the present invention the following terms are defined:

[0049] • The term "comprising" means including, but it is not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0050] • The term "consisting of’ means including, and it is limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0051] • The term “pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included with the pharmaceutical composition of the invention and that causes no significant adverse toxicological effects to the patient. • The expression “proteins that have been co-translationally or post-translationally modified trough the addition of nucleotide sugars” refers to proteins to which sugar residues have added during or after translation. These modifications can be detected using antibodies that recognize the specific nucleotide sugars in the form in that they are bound to the modified proteins. For instance, the O-GlcNAc antibodies recognize GlcNAc only when it has been added to a protein through O-linked glycosylation. Glycosylation is often characterized as a post-translational modification. While this is true with other types of glycosylation, N-glycosylation often occurs co-translationally, in that the glycan is attached to the nascent protein as it is being translated and transported into the ER. This is the reason why the claim mentions “co-translationally or post-translationally”.

[0052] • The expression “N-linked glycosylated proteins or O-linked glycosylated proteins, preferably O-linked N-acetylglucosamine acylated (O-GlcNAcylated)” refers to proteins to which a nucleotide sugar has been added either through N-linked or O-linked glycosylation. For instance, O-linked N-acetylglucosamine acylated (O-GlcNAcylated) proteins are proteins to which an GlcNAc residue has been added through O-linked glycosylation.

[0053] Description of the figures

[0054] Figure 1. Hexosamine biosynthetic pathway is increased in Marfan mouse aortas. A) Heatmap showing HBP-related genes from RNAseq performed in aortas from 24weeks-old Fbnl+ / +and Fbnlcl039G / +mice. B) Relative mRNA levels analyzed by q-PCR, of HBP-related genes of aortic extracts from 24weeks-old Fbnl+ / +and Fbnlcl039G. C) Relative G^?t2-mRNA levels from aortic extracts from young Fbnl+ / +and Fbn 1C1O39G / +mice. D) Representative immunoblot of O-glycosylated proteins and Gfpt2 of aortic extracts from 24weeks-old Fbnl+ / +and FbnlCI039G / +mice. E) Representative confocal-immunofluorescence analysis for HBP related proteins and O-glycosylated proteins (Red), Elastin (Green) and Dapi (Blue). F) Representative confocal-immunofluorescence analysis for O / N acetyl-glucosilated proteins stained with wheat germ agglutinin (WGA), smooth muscle actin (Red) as marcker for VSMCs. G) N-acetyl- galactosamine and N-acetylglucosamine (NAcHex) plasmatic levels from 24weeks-old Fbnl+ / +and FbnlCI039G / +mice. H) Representative immunoblot of O-glycosylated proteins from plasma of Fbnl+ / +and FbnlCI039G / +mice. I) Representative immunoblot of O- glycosylated proteins, Gfpt2 and UAP1 from primary VSMCs silenced for Fbnl for 5 days. Figure 2. Boosting the hexosamine biosynthetic pathway is deleterious for aortic homeostasis in Fbnl+ / +and FbnlCI039G / +mice. A) Scheme depicting the experimental approach for panels B-E. Glucosamine (Glen) was administrated by drinking water (5%). B) Representative ultrasound images of ascending aorta at the end of the experiment. C) Aortic diameters along the experiment. D) Systolic blood pressure along the experiment. E) Representative histological analysis of elastin (EVG, in black), proteoglycan accumulation (Alcian blue, blue) in the cohort of mice from A-D panels. F) Quantification of elastin breaks in the cohort of mice from A-D panels. G) Lentiviral transduction of GFPT2 overexpressing viruses in primary murine VSMCs. Representative immunoblots of GFPT2 and O-glycosylated proteins.

[0055] Figure 3. Pharmaceutical inhibition of the hexosamine biosynthetic pathway improves aortic homeostasis in FbnlCI039G / +mice. A) Representative immunoblot analysis of VSMCs Fbnl- silenced, or sh-control incubated with 6-diazo-5-oxonorleucine (GFPT -inhibitor DON, lOnM) for 48h. B) Flow-cytometry analysis of O-glycosylated and O / N-Nacetylglycosilated proteins in VSMCs control or F / w / -silenced treated w / o DON. C) Relative mRNA levels of typical Marfan upregulated genes from VSMCs Fbnl -silenced 1 incubated w / o DON. D) Scheme depicting the experimental approach for panels E-I. Mice age was 20weeks-old, DON was administered by osmotic minipumps (5ng / Kg / day). E) Representative ultrasound images of ascending aorta at the end of the experiment. F) Aortic and systolic blood pressure along the experiment. G) Representative histological analysis and quantification of elastin (EVG, in black), proteoglycan accumulation (Alcian blue, blue) in the cohort of mice from A-D panels. H) Representative immunoblot of O-glycosylated proteins from aortic extracts in the same cohort of mice showed in panels D-G. I) Representative confocal-immunostaining analysis of O-glycosylated proteins (O-GlcNac) and N / o-acetylglucosylated proteins (WGA); SMA was used as control for VSMCs.

[0056] Figure 4. Pharmaceutical inhibition of Hexosamine biosynthetic pathway reduces Integrative Stress Response (IRS) in Fbnlcl039G / +mice. A) Heatmap showing IRS-related genes from RNAseq performed in aortas form the cohort of mice showed in Fig. 1. B) Representative immunoblot analysis and C) qPCR analysis, IRS-markers from VSMCs F / w / -silenced incubated w / o DON for 48h and D) representative immunoblot analysis of IRS-markers and E) qPCR analysis, from VSMCs transduced with lentivectors that overexpress GFPT2. F) Representative confocal-immunostaining analysis of IRS-markers in the same cohort of mice showed in Fig3. G) Representative immunoblot analysis and H) qPCR analysis, of IRS- markers from aortic extracts in the same cohort of mice showed in Fig3. 1) Heatmap of median reads showing IRS-related genes from RNAseq performed in aortas form the cohort of mice showed in Fig.3 (n=4). J) Heatmap of median reads showing synthetic VSMCs-phenotype genes from RNAseq performed in aortas form the cohort of mice showed in Fig.3 (n=4).

[0057] Figure 5. Pharmaceutical inhibition Integrative Stress Response reduces aortic aneurysm in FbnlC1039G / + mice. A) qPCR analysis of RS-markers from VSMCs Fbnl -silenced 1 incubated w / o DON for 48h and B) Scheme depicting experimental approach for panel C. Mice age was 20weeks-old, ISRIB (IRS-Inhibitor) was administrated by osmotic minipumps (lOmg / Kg / day). C) Aortic and Systolic blood pressure along the experiment.

[0058] Figure 6. Aortic sections from MFS-patients show an increase of HBP and IRS pathways in aortic. Representative confocal-immunostaining analysis of O-glycosylated proteins (O- GlcNac) and N / O-acetylglucosylated proteins (WGA); GFPT2 and p-PERK in aortic media of control and MFS-patients SMA was used as control for VSMCs.

[0059] Figure 7. The HBS and IRS are increased in a murine model of atherosclerotic-abdominal aortic aneurysm (AAA). A) Scheme of the experiment, ApoE null mice were fed with high cholesterol and fat diet (western-diet) 21 -days prior Angll infusion. Abdominal aortic diameter after Angll infusion. B) Relative mRNA levels analysed by qPCR of HBS and IRS markers in ApoE~GAAA mice and ApoE~Gcontrols (without Angll and western diet).

[0060] Figure 8. The HBP genes are upregulated in the aortas of a murine model MFS. (A) Scheme depicting the HBP role in O-glycosylation, N-linked glycosylation and GAGs. (B) Gene expression heatmap of HBP, O-glycosylation, N-linked glycosylation and GAGs-related genes from RNA-sequencing analysis of aortic medial tissue from 24 weeks old Fbnl+'+and from FbnlCI04IG / +mice (n=4). (C) Quantitative reverse transcription polymerase chain reaction analysis of Gjpt2, Uapl, Gnpatl, Mgat2, Ogt, Oga mRNA relative expression in aortic extracts from 20 / 24-week-old FbnlCI04IG / +and Fbnl+I+mice. (D) Quantitative reverse transcription polymerase chain reaction analysis of Gfpt2 mRNA relative expression in aortic extracts from 4- or 8-weeks-old FbnlCI04IG / +and / ’7w / mice. (E) Representative confocal imaging of Gfpt2, Uapl, O-GlcNac (red); elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in the ascending aorta from mice

[0061] (n=6). (F) Representative immunoblot analysis of O-GlcNac proteins, Gfpt2 in aortic extracts from 20-week-old FbnlCI04IG / +and Fbnl+I+mice (n=6). Actin was used as loading control. (G) Representative confocal imaging of WGA (gray), Smooth muscle actin (Sma, red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in the ascending aorta from 20- week-old FZw7c;cwG / +and FZw7+ / +mice (n=6). (H) Representative immunoblot analysis of Gfpt2, Uapl, O-GlcNac proteins in extracts from primary murine VSMCs transduced with shFbnl or shScr (ShControl) for 5 days (n=3). Actin was used as loading control. Data are mean±SEM. Statistical significance was assessed by Student t test. *F<0.05, **F<0.01 mice.

[0062] Figure 9. Boosting HBP induces aortic dilatation and medial degeneration. (A) Experimental design, panels A-E, 20-weeks old FbnlCI04IG / +and Fbnl+I+mice were with or without glucosamine (GlcN) in driking water for 28 days (B) Representative aortic ultrasound images after 28 days of control or GlcN treatment. Discontinuous red lines mark the lumen boundary, scale bar 1mm. (C) Evolution of maximal AsAo and AbAo diameter. (D) Representative histologic staining with EVG and Alcian blue in the AsAo and quantification of elastin breaks (E). (F) Representative confocal imaging of O-GlcNac proteins (red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in the ascending aorta from Fbnl+'+mice treated with or without GlcN for 28 days (n=6). (G) Representative confocal imaging of WGA (gray), Smooth muscle actin (Sma, red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in the ascending aorta from Fbnl+'+mice treated with / without GlcN for 28 days (n=6). (H) Representative immunoblot analysis of Gfpt2 and O-GlcNac proteins in LV-Mock (control) or Lv-GFPT2 transduced primary murine VSMCs for 5 days (n=3). (I) Experimental design, panels J-M, 12-weeks old C57BL / 6 wild-type mice were injected with LV-Mock or Lv-GFPT2 lentivectors for 28 days. (J) Evolution of maximal AsAo diameter after inoculation of LV-Mock (control) or Lv-GFPT2 lentiviral vectors. (K) Representative confocal imaging GFP or GFPT2 (red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in ascending aortas from mice after 30 days of inoculation of LV- Mock (control) or Lv-GFPT2 lentivectors (n=6). (L) Representative confocal imaging of WGA (gray), O-GlcNac proteins (red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in ascending aortas from mice after 30 days of inoculation of LV-Mock (control) or Lv- GFPT2 lentivectors (n=6). (M) Representative histologic staining with EVG and Alcian blue in the AsAo and quantification of elastin breaks in ascending aortas from mice after 30 days of inoculation of LV-Mock (control) or Lv-GFPT2 lentivectors. Data are mean±SEM. Statistical significance was assessed by 2-way repeated measurements ANOVA (C,J), by 1-way ANOVA (E) or t-Test (M). *F<0.05, **F<0.01, ***F<0.001, ****F<0.0001 vs Control. Figure 10. HBP inhibition by DON restores aortic dilatation and medial degeneration in MFS mice. (A) Representative flow cytometry histograms and statistical analysis of O- GlcNAc and WGA staining of primary VSMCs from FbnlCI04IG / +and F 'bnl+'+mice treated with or without DON for 24h. (B) Experimental design, panels B-J, 20 / 22-weeks old FbnlCI04IG / +and Fbnl+I+mice were infused with minipumps with saline (control) or DON for 28 days. (C) Representative aortic ultrasound images after 28 days of control or DON treatment. Discontinuous red lines mark the lumen boundary, scale bar 1mm. (D) Evolution of maximal AsAo and AbAo diameter. (E) Representative histologic staining with EVG and Alcian blue in the AsAo and quantification of elastin breaks and aortic medial thickness (F) (n=6). (G) Representative confocal imaging of O-GlcNac proteins or SMA (red), WGA (gray), elastin (green, autofluorescence), DAPI-stained nuclei (blue) in the ascending aorta from Fbnl+,+and Fbnlcl041G / +mice treated with / without DON for 28 days (n=6). (H) Representative immunoblot analysis and quantification of O-GlcNac proteins in aortic extracts from Fbnlcl041G / +and Fbnl mice with saline or DON for 28 days (n=4). (I) GAGs serum levels, N-acetylGlucosamine / N-acetyl Galactosamine and GlcN levels in sera from 20-week- o\< FbnlG1041G / +and mice with saline or DON for 28 days. Data are mean±SEM . Statistical significance was assessed by 1-way ANOVA (A,F,H,I) or 2-way repeated measurements #P<0.05, ## <

[0063] Figure 11. PGM3 inhibition by FR054 decreases aortic dilatation and glycans in MFS aortas. (A) Representative flow cytometry histograms and statistical analysis of O-GlcNAc and WGA staining of primary VSMCs from FbnlC1041G / + and Fbnl+ / + mice treated with or without FR054 lOOpM for 48h. (B) Quantitative reverse transcription polymerase chain reaction analysis of Sppl and Fgf2 mRNA relative expression in extracts from VSMCs of FbnlC1041G / + and Fbnl+ / + mice mice treated with or without FR054 100 pM for 48h. (C) Experimental design, panels D-H, 18 / 20-weeks old FbnlC1041G / + and Fbnl+ / + mice were infused with minipumps with saline (control) or FR054 for 28 days. (D) Evolution of maximal AsAo and AbAo diameter. (E) Representative histologic staining with EVG and Alcian blue in the AsAo and quantification of elastin breaks. (F) Representative confocal imaging of O- GlcNac proteins or SMA (red), WGA (gray), elastin (green, autofluorescence), DAPI-stained nuclei (blue) in the ascending aorta from Fbnl+ / + and FbnlC1041G / + mice treated with / without DON for 28 days (n=6). (G) GAGs serum levels from FbnlC1041G / + and Fbnl+ / + mice with saline or FR054 for 28 days. (H) FR054 treatment for 28 days, reduced the Col lai and Klf4 mRNA levels in aortas from FbnlC1041G / + mice. Notably, FR054 treatment in FbnlC1041G / + mice (Figure 1 lH).Data are mean±SEM . Statistical significance was assessed by 1-way ANOVA (A,F,H,I) or 2-way repeated measurements ANOVA (D). *P<0.05, **P<0.01, ***P<0.001, for FbnlC1041G / + vs Fbnl+ / +; #P<0.05, ##P<0.01, ####P<0.0001 for Fbn 1 C 1041 G / + DON vs Fbn 1 C 1041 G / +.

[0064] Figure 12. ISR is activated by HBP in aortic tissue. (A) Gene expression heatmap of ISR- related genes from RNA-sequencing analysis of aortic medial tissue from 24-weeks old Fbnl+I+mice (n=4) and from FbnlCI04IG / +mice (n=4). (B) Representative confocal imaging p- Perk, p-eIF2a or atf4(red); elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in ascending aortas from 20-week-old FbnlCI04IG / +and Fbnl+I+mice infused with saline or DON for 28 days (n=5). (C) Representative immunoblot analysis and quantification of O- GlcNac proteins, p-Perk P-eIF2a or Atf4 in aortic extracts from 20-week- old FbnlCI04IG / +arA Fbnl mice infused with saline or DON for 28 days. (D) Quantitative reverse transcription polymerase chain reaction analysis of Atf4, Atf6 and Tgfb2 mRNA relative expression in aortic extracts from 20 / 24-week-old FbnlCI04IG / +and Fbnl+I+mice treated for 28 days with DON. (E) Gene expression heatmap of ISR- and extracellular matrix-related genes from RNA-sequencing analysis of aortic medial tissue from 20 weeks old Fbnl+'+mice (n=4) and from FbnlCI04IG / +mice infused with saline or DON for 28 days (n=4). (F) Representative confocal imaging p-Perk, p-eIF2a or Atf4(red); elastin (green, autofluorescence), and DAPI- stained nuclei (blue) in ascending aortas from 20 / 22 weeks- / ’7w / mice treated with or without GlcN for 28 days (n=6). (G) Representative confocal imaging p-Perk, P-eIF2a or atf4(red); elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in ascending aortas from 20-week-old Fbnl+'+mice transduced with LV-Mock or LV-GPFT2 for 30 days (n=5). Data are mean±SEM . Statistical significance was assessed by 1-way ANOVA

[0065] Figure 13. ISR contributes to VSMCs-dysfunction and aortic pathology in MFS-mice. (A) Representative confocal imaging of Scarlet fluorescence (red) in primary VSMCs from Fbnlcl041G / +and Fbnl+l+mice transduced with LV-ATF4 Scarlet after 3 days treated with Vehicle, DON or ISRIB for 48 hours (n=3). (B) Representative confocal imaging of puromycinylated proteins (green) and smooth muscle actin Sma (Red), in primary VSMCs from FbnlCI04IG / +and Fbnl+I+mice incubated with puromycin for 5 minutes and treated with vehicle, DON or ISRIB for 48 hours (n=4). (C) Experimental design, panels A-E, 20 / 22-weeks old FbnlCI04IG / +and Fbnl+I+mice were infused with vehicle or ISRIB in minipumps for 28 days. (D) Evolution of maximal ascending (AsAo) and abdominal (AbAo) diameter. (E) Representative aortic ultrasound images after 28 days of control or ISRIB treatment. Discontinuous red lines mark the lumen boundary, scale bar 1mm. (F) Representative histologic staining with EVG and Alcian blue in the AsAo and quantification of elastin breaks after 28 days of vehicle or ISRIB infusion (G). (H) Representative confocal imaging of Atf4 (red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in the ascending aorta from and Fbnlcl039G / +mice treated with / without ISRIB for 28 days (n=4). (I) Representative immunoblot analysis and quantification of Atf4 in aortic extracts from FbnlCI04IG / +and Fbnl+I+mice with vehicle or ISRIB for 28 days. (J) Quantitative reverse transcription polymerase chain reaction analysis oiAt 4, Atf6 and Tgfb2 mRNA relative expression in aortic extracts from FbnlCI04IG / +and Fbnl+I+mice infused with vehicle or ISRIB for 28 days. Data are mean±SEM Statistical significance was assessed by 2-way repeated measurements ANOVA (B) or 1-way ANOVA (G,J,I) or ** <0.01, *** <0.001, for Fbnlcl041G / +vehicle vehicle ; # <0.05, ## <0.01, ####P<0.0001 for FbnlCI04IG / +ISRIB vs FbnlCI04IG / +Vehicle.

[0066] Figure 14. HBP-ISR axis is upregulated in MFS patients. (A) Representative confocal immunostainings and quantification (B) of O-GlcNac (Red) and WGA (Gray) elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in medial layer of aortic sections from control donors or patients with MFS (n=8; n=4 females, n=4 males; per group). (C) GAGs serum and plasmatic levels, in two different cohort of patients. (D) N-acetylGlucosamine / N- acetyl Galactosamine (GlcNac / GalNac) and Glucosamine (GlcN) levels in plasma from Cohort-2 of control or MFS-patients. (E) Representative confocal immunostainings. (F) quantification of GFPT2, p-PERK, p-EIF2a, ATF4 (red), elastin (green, autofluorescence), and DAPI-stained nuclei (blue) in medial layer of aortic sections from control donors or patients with MFS (n=8; n=4 females, n=4 males; per group). (G) Pharmacological Inhibition of HBP by DON / FR054 or ISR by ISRIB reverses aortic aneurysm in Marfan mice. Circulating HBP metabolites and glycosaminoglycans operate as biomarkers of Marfan Syndrome patients and mice. The metabolic HBP-ISR axis might be targeted for treating thoracic aortic-related disorders. Data are mean±SEM, statistical significance was assessed by Student / test. * <0.05, **P<0.01, *** <0.01 **** <0.0001 vs Control.

[0067] Detailed description of the invention The present invention is illustrated by means of the Examples set below without the intention of limiting its scope of protection.

[0068] PHASE I

[0069] Example 1. Material and Methods

[0070] Example 1.1. Animal Procedures

[0071] Marfan mouse model which harbor a Fbnlcl039G / +allele (JAX stock #012885). Wild-type littermates were used as controls for Marfan mice. DON dissolved in saline, (Sigma-Aldrich), at 5 ng* kg1min1and ISRIB (MedChemExpress) at lOmg* kg1min1were infused using subcutaneous osmotic minipumps (Model 2004, Alzet Corp). NR (Novalix) was administered intraperitoneally at 1000 mg / kg in saline every other day. Mice were housed at the pathogen- free animal facility of the CNIC and CBMSO following the animal care standards of the institution. Animal procedures were approved by the CNIC and CBMSO-UAM Ethics Committee and the Madrid regional authorities and conformed with EU Directive 2010 / 63EU and Recommendation 2007 / 526 / EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201 / 2005.

[0072] The ApoE (Apolipoprotein E)-deficient mice, B6.129P2-ApoetmlUnc / J (JAX stock No. 002052), in pure C57 / BL6J background were obtained from Charles Rivers. To accelerate atherosclerosis and increase the rate of aortic ruptures, 12-week-old male mice were fed a western diet (high fat and cholesterol diet, SSNIFF-S9167-E011, Ssniff Spezialdiaten, Germany), 3 weeks prior and maintained to Ang-II (angiotensin-II) infusion. Only male mice were studied as females are protected from developing AAA. Ang-II was dissolved in saline (Sigma- Aldrich) and infused at 1 pg / kg min using subcutaneous osmotic mini pumps (Model 2004, Alzet Corp.).

[0073] Example 1.2. Blood pressure measurements and in vivo imaging

[0074] Arterial blood pressure (BP) was measured by the mouse-tail cuff method using the automated BP-2000 Blood Pressure Analysis System (Visitech Systems, Apex, NC, USA). Mice were trained for BP measurements every day for five consecutive days. After training period, BP was measured one day before treatment to determine the baseline BP values in each mouse cohort. BP measurements were recorded in mice located in a tail-cuff restrainer over a warmed surface (37 °C). Fifteen consecutive systolic and diastolic BP measurements were made, and the last 10 readings per mouse were recorded and averaged. For in vivo ultrasound images, the aortic diameter was monitored in isoflurane-anesthetized mice (2% isoflurane) by high- frequency ultrasound with a VEVO 2100 echography device (VisualSonics, Toronto, Canada) at 30-micron resolution. Maximal internal aortic diameters were measured at diastole using VEVO 2100 software, version 1.5.0.

[0075] Example 1.3. Cell procedures

[0076] Isolation and culture of primary mouse VSMCs were previously described. Tissue was digested with a solution of collagenase and elastase (Worthington Biochem) until a single-cell suspension was obtained. All experiments with primary VSMCs were performed during passages 2-7. Lentiviral transduction was performed in VSMCs over 5 h at a multiplicity of infection = 3. The medium was then replaced with fresh DMEM supplemented with 10% FBS and cells were cultured for five more days, treated with NR for five more days and serum- starved for 16h. The HEK-293T (CRL-1573) and Jurkat (Clone E6-1, TIB-152) cell lines, required for high-titer lentivirus production and lentivirus titration respectively, were purchased from ATCC. The experiments were performed during passages 5-10. All cells tested negative for Mycoplasma.

[0077] Example 1.4. Lentivirus production and infection

[0078] The GFPT2 and GFP coding sequence was obtained from Origene. Lenti viruses expressing shRNA targeting murine Fbnl, and control shRNA were purchased from Sigma-Aldrich. Pseudo-typed lentiviruses were produced by transient calcium phosphate transfection of HEK- 293T cells and concentrated from culture supernatant by ultracentrifugation (2 hours at 128,000xg; Ultraclear Tubes; SW28 rotor and Optima L-100 XP Ultracentrifuge; Beckman). Viruses were suspended in cold sterile PBS and titrated by transduction of Jurkat cells for 48h. Transduction efficiency (GFP-expressing cells and Puromycin resistant-cells) and cell death (propidium iodide staining) were quantified by flow cytometry.

[0079] Example 1.5. Real-time and quantitative PCR

[0080] Aortas were extracted after perfusion with 5 ml saline solution, and the adventitia layer was discarded. Liquid nitrogen frozen tissue was homogenized using a cold-mortar and an automatic bead homogenizer (MagNA Lyzer, Roche). Total RNA was isolated with Trizol (Life Technologies). Total RNA (1 pg) was first digested with DNAse and reverse-transcribed with Maxima First Strand cDNA Synthesis Kit (ThermoFisher). For analysis of mtDNA levels, total DNA from cells and tissues was extracted with the SurePrep kit (Fisher Scientific) or Trizol respectively, according to the manufacture’ s guidelines. qPCR reactions were performed in triplicate with SYBR master mix (Promega), according to the manufacturer’s guidelines. To examine probe specificity, we conducted a post-amplification melting-curve analysis. For each reaction, only one melting-temperature (Tm) peak was produced. The amount of target mRNA in samples was estimated by the 2-CT relative quantification method, using B2M, YWHAZ and PP1A for normalization. Fold ratios were calculated relative to mRNA expression levels from controls.

[0081] Example 1.6. Library preparation and Illumina sequencing

[0082] Aortas were extracted after perfusion of cold saline solution and the adventitia layer was discarded. Frozen tissue was homogenized, and total RNA was isolated with Trizol (Roche). RNA from Libraries were prepared according to the instructions of the Kit “NEBNext Ultra Directional RNA Library Prep kit for Illumina” (New England Biolabs), following the protocol “Poly(A) mRNA Magnetic Isolation Module”. The input yield of total RNA to start the protocol was >300 ng quantified by an Agilent 2100 Bioanalyzer using a RNA 6000 nano LabChip kit. The obtained libraries were validated and quantified by an Agilent 2100 Bioanalyzer using a DNA7500 LabChip kit and an equimolecular pool of libraries was titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit forLightCycler480” (Kapa BioSystems) and a reference standard for quantification. After processing on the Illumina HiSeq 2500 instrument, FastQ files were generated containing nucleotide data and quality scores for each position. RNA-seq reads were mapped to the Mus musculus reference genome, GRCm38.p6, using Hisat2 v2.1.0 software. Reads were then preprocessed with SAMtools vl.7 to transform SAM files into BAM files, and sorted. These files were used as an input for the HTSeq vO.6.1 package, that produces for each sample a file containing the mapped reads per gene (as defined by the Mus musculus, GRCm38.96 version, gff file). Once obtained HTSeq read counts, the Bioconductor RNA-Seq workflow was followed to detect the expression differences of genes using the DESeq2 statistical package. Ingenuity Pathway Analysis (IP A) was used to identify cellular biological functions, gene clusters and upstream regulators.

[0083] Example 1.7. Immunoblot

[0084] For Western blot (WB) analysis, cells were lysed at 4 °C in RIPA buffer containing protease and phosphatase inhibitors cocktail (Sigma). Proteins were separated by SDS-PAGE and transferred onto 0.45 pm pore size Immobilon PVDF membrane (Millipore). PVDF membranes were blocked with TBS-T (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% (w / v) milk. Membranes were incubated with primary antibodies diluted 1 / 500 to 1 / 1000 followed by TBS-T washes and incubation with HRP-conjugated secondary antibodies (GE healthcare). The signal was visualized by enhanced chemiluminescence with Luminata Forte Western HRP Substrate (Millipore) and the ImageQuant LAS 4000 imaging system. The following antibodies were used: anti-O-GlcNac RL2 monoclonal (Abeam), Anti- GFPT2 (Proteintech), anti -U API (Novus) anti -PERK (Thermo), and anti-pEIF2 and ATF4 (Cell signalling), anti Actin (Abeam) and anti-a-tubulin (Cell Signaling).

[0085] Example 1.8. Aortic histology

[0086] After euthanization by CO2 inhalation, mice were perfused with saline. Aortas were then isolated and fixed in 10% formalin overnight at 4 °C. Paraffin cross-sections (5pm) from fixed organs were stained with Masson-Tri chrome, Alcian blue or Verhoeff elastic-van Gieson (EVG), or they were used for immunohistochemistry or immunofluorescence. Elastic fibers were stained with a modified Verhoeff Van Gieson elastin stain kit (Sigma-Aldrich). Elastic lamina breaks, defined as interruptions in the elastic fibers, were counted in the entire medial layer of three consecutive cross-sections per mouse, using 4-16 mice per experiment, and the mean number of breaks was calculated. For immunostaining, the deparaffinized sections were rehydrated, boiled to retrieve antigens (10 mM citrate buffer, Triton-x 0,05%, pH 6) and blocked for 45 min with 10% goat normal serum, 5% horse serum, Triton-x 0,05% and 2% BSA in PBS. Samples were incubated with the following antibodies for immunohistochemistry or immunofluorescence: monoclonal anti-SMA (1 / 500, C6198, Sigma), WGA-647 (1 / 2.000 invitrogene) polyclonal anti-GFPT2 (1 / 300, Proteintech), monoclonal anti-O-GlcNac (1 / 300, Abeam), polyclonal anti-p-PERK (Thermo) and anti-pEIF2, ATF4 (1 / 50 Cell Signaling). Specificity was determined by substituting primary antibody with unrelated IgG (Santa Cruz). For immunohistochemistry, endogenous peroxidase and biotin was blocked with 1% H2O2- methanol for 10 minutes and with biotin-block kit (Vector Laboratories) respectively. Color was developed in all samples at the same time with DAB (Vector Laboratories), and sections were counterstained with hematoxylin and mounted in DPX (Fluka). For immunofluorescence, secondary antibodies were Alexa-Fluor-546-conjugated goat anti-rabbit and Alexa-Fluor-647- conjugated goat anti-rabbit (BD Pharmigen). For F-actin determination, fixed aortas were embedded in OCT (Tissue-Tek Sakura), 5pm cross-sections were incubated for 30 minutes with 1 : 1000 Phalloidin-657 (Millipore) after 10 min of incubation with 0.3% Triton X-100 in PBS. Sections were mounted with DAPI in Citifluor AF4 mounting medium (Aname). Images were acquired at 1024 x 1024 pixels, 8 bits, using a Zeiss-LSM800 microscope with 40 oilimmersion objectives.

[0087] Example 1.9. Human samples

[0088] The study was approved by the ethics and clinical research committee of Institute de Salud Carlos III and Cantabria University. Aortas for use as controls were obtained anonymously from multiorgan transplant donors after written informed consent was obtained from their families. During preparation of the heart for transplantation, excess ascending aortic tissue was harvested for the study. Samples from patients were obtained during elective or emergency aortic root surgery for aortic aneurysm-dissection. Patient clinical data were retrieved while maintaining anonymity. Tissues were immediately fixed, kept at room temperature for 48 h and embedded in paraffin. DNA and RNA extraction from paraffin sections were performed with All Prep DNA / RNA FFPE Kit (Quiagen).

[0089] Example 1.10. Statistical analysis

[0090] Normality of data was checked using Shapiro-Wilk test. F-test was used to assess the equality of variance assumption. Differences between two groups were assessed using unpaired Student’s t-test, t-test with Welch's correction for unequal variances, or Mann-Whitney U test where appropriate. Differences in experiments with >3 groups were analyzed by one-way, two- way, repeated-measurements two-way analysis of variance (ANOVA) or mixed-effects linear model and Newman’ spost hoc test, as appropriate. For the statistical analysis of RNA-seq data the p-values are corrected using the FDR method, (FDR <0.05). For survival curves, differences were analyzed with the log-rank (Mantel-Cox) test. Multiple linear regression analysis adjusted for age was performed with R software. For all other analysis GraphPad Prism software 9 was used. Statistical significance was indicated as *P < 0.05, **P < 0.01, ***p < 0.001 and ****p < 0.0001. Sample size was chosen empirically based on our previous experiences in the calculation of experimental variability; no statistical method was used to predetermine sample size. Before data analysis, outliers were identified and excluded by using the ROUT method (Q value =5%) to identify outliers provided with GraphPad Prism 9.

[0091] The numbers of animals used are described in the corresponding figure legends. All experiments were done with at least three biological replicates. Experimental groups were balanced in terms of animal age, sex and weight. Animals were genotyped before experiments, and they were all caged together and treated in the same way. Sex and age are indicated in the figure legends. Appropriate tests were chosen according to the data distribution. Variance was comparable between groups in experiments described throughout the manuscript. For the rest of experiments, no randomization was used to allocate animals to experimental groups, and investigators were not blinded to group allocation during experiments or to outcome assessments.

[0092] Example 2. Results

[0093] Example 2.1. Hexosamine biosynthetic pathway is increased in aortas from a murine model of MFS

[0094] The inventors performed a transcriptional analysis by next generation sequencing to unveil an intermediate stage of aortic disease in the aortas of 24-week-old Fbnlcl039G / +mice, which reproduce the syndromic phenotype, aortic dilatation, aneurysms, and coronary microvascular dysfunction (CMD) seen in MFS patients. By using Ingenuity Pathway Analysis, they identified that the hexosamine biosynthetic pathway is increased in MFS-mice aortas (p-value= 0,047). The HBP is a minor branch of glycolysis, accounting for only 2-5% of total glucose metabolism in homeostasis. The HBP final metabolite is the nucleotide sugar UDP-N- acetylglucosamine (UDP-GlcNAc). This sugar is used to modify intracellular proteins co- translationally or post-translationally (N- or O-glycosylation) or for the synthesis and assembly of glycosaminoglycans (GAGs), proteoglycans (PGs) and glycolipids. The inducible ratelimiting enzyme of HBP, Glutamine-fructose-6-phosphate transaminase 2 (GJpt2 and the downstream enzymes Uapl (UDP-N-Acetylglucosamine Pyrophosphorylase 1) and Gnpatl (glucosamine-phosphate N-acetyltransferase 1) were significantly increased in MFS-aortas (Fig. 1A). The pair of enzymes O-GlcNAc transferase (Ogf) and O-GlcNAcase (Oga), which are involved in O-GlcNAcylation, were upregulated in MFS aortas (Fig. 1A). These two enzymes mediate the dynamic cycling of O-GlcNAc on a wide array of cytosolic, nuclear, and mitochondrial proteins. Moreover, the inventors found that Golgi-enzymes involved in N- glycosylation such as Mgatl, Mgat2, Mgat4a, Mgat5b (Alpha- 1,3-Mannosyl-Glycoprotein 2- Beta-N-Acetylglucosaminyltransferase 1, 2, 4a, 5b); were upregulated in MFS-aortas (Fig. 1A). Transcripts of Gne (Glucosamine UDP-N-Acetyl-2-Epimerase / N-Acetylmannosamine Kinase), which initiates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acid; B3gnt3 (UDP-GlcNAc: BetaGai Beta-1,3-N-Acetylglucosaminyltransferase 3), which is involved in the biosynthesis of poly-N-acetyllactosamine chains and the biosynthesis of the backbone structure Sialyl Lewisx(CD15s); and Hasl / Has3 (hyaluronan synthase 1 / 3), which synthesizes hyaluronic acid, were increased in MFS-aortas (Fig. 1A). These data were confirmed by qPCR analysis in aortic extracts from MFS-mice (Fig. IB). The inventors performed an analysis of mRNA Gfpt2 aortic expression at different ages. Gfpt2 mRNA levels are significantly increased in the very onset of aortopathy at 4weeks-old in Fbnlcl039G / +mice when they show barely aortic phenotype (Fig. 1C).

[0095] By immunoblot analysis the inventors found that the GFP2 and O-GlcNAc protein levels were increased in aortic samples of MFS-mice (Fig. ID).

[0096] These data point to an increase of the HBP and downstream enzymes, which might drive an increase of O-GlcNAc, N-glycosylation and GAGs levels in aortas from MFS mice. The inventors assessed HBP-levels by histological analysis of Gfpt2, Uapl, O-GlcNac by RL2 monoclonal antibody (Fig. IE) and extracellular N-glycosylation levels by fluorescent-WGA (Wheat germ agglutinin) (Fig.l G). WGA is a lectin that selectively binds to N- acetylglucosamine residues, mainly present in the ECM. Moreover, in blood plasma from MFS-mice, N-acetylhexosamine and O-GlcNAc proteins are increased (Fig.l G, H).

[0097] To model MFS in vitro, the inventors silenced Fbnl with lentiviral vectors in primary murine VSMCs. Validating the transcriptomic data from MFS mice, shFbnl VSMCs displayed increased mRNA and protein levels of Gfpt2 and Uapl protein levels (Fig. II). Moreover, shFbnl VSMCs showed an increased levels of O-GlcNac and WGA staining (Fig. II).

[0098] These data support that aortas from Fbnlcl039G / +mice or Fbnl -deficient VSMCs present augmented HBP that rise O-GlcNac and N-glycosylation levels in aortic tissue and N- acetylhexosamine blood plasmatic levels.

[0099] Example 2.2 HBP promotes aortic dilation and medial degeneration in wild type- and MFS- mice

[0100] Because ground substance accumulation in CMD is a characteristic of TAAD the inventors hypothesized that an excessive HBP-activity might be involved in the increase of PGs and GAGs that forms the ground-mucoid substance during TAAD development. The inventors treated mice with glucosamine to fuel HBP. Glucosamine enters the cell, it is transformed to glucosamine-6-phosphate and finally, is converted to UDP-GlcNAc. Glucosamine treatment in drinking water in wild-type mice (Fbnl ) for 28 days significantly increased aortic diameter and decreased systolic blood pressure (BP) after 7 days of treatment (Fig. 2A-D). Importantly, glucosamine administration in Fbnlcl039G / +mice increased aortic diameter compared with control FbnlCI039G / +mice (Fig. 2A-D). Glucosamine reproduced histological features of CMD in Fbnl+ / +mice and worsen in FbnlCI039G / +mice aortic architecture such as elastin fiber fragmentation and amorphous matrix accumulation in Alcian blue staining after 28 days of treatment (Fig. 2E-F). Lentiviral transduction with GFPT2 increases O-GlcNAc proteins (Fig. 2G)

[0101] These results indicate that an excessive HBP activity is involved in accumulation of ground substance in CMD during TAAD development and is detrimental for aortic homeostasis.

[0102] Example 2.3. HBP has critical role in the aortopathy of MFS

[0103] To investigate the therapeutic potential of HBP inhibitors in TAAD, the inventors treated shF7w / -VSMCs with DON (6-diazo-5-oxo-L-norleucine) for 48h. DON inhibits HBP by blocking GFPT enzymatic activity. DON treatment efficiently reduced O-GlcNac in AxFbril- VSMCs (Fig. 3A, B). DON incubation decreased mRNA levels of classical MFS genes such as Tgbl, Fgf2 growth factors, osteopontin (.s / yi / ) and colagen 1 (Col lai (Fig. 3C). The inventors next tested the therapeutic potential of DON for 28-days in the development of aneurysm in MFS mice by modulating HBP-activity (Fig. 3D). Aortic dilation and blood pressure were completely normalized after 7 days of treatment in 24-weeks FbnlCI039G / +mice (Fig. 3E, F). Moreover, DON-treatment restored histologic features of aortic degeneration in MFS, such as medial thickening, elastic fiber fragmentation, GAG and PG deposition by Alcian blue staining after 28 days of DON-treatment (Fig. 3G). DON treatment for 28 days reduces O-GlcNac levels and WGA staining in aortic tissue (Fig. 3H, I).

[0104] These data indicate that HBP-inhibition declines O-GlcNac and WGA staining, hence, decreasing CMD, improving aortic architecture and homeostasis in an MFS mice model.

[0105] Example 2.4. IRS is induced by HBS in MFS

[0106] Analyzing RNA-seq from aortas of 24-week-old MFS-mice, the inventors found that endoplasmic reticulum (ER) stress is activated (p-value 0.032, z-score 1,342). In MFS-mice aortas gene expression of ER-stress profile genes such as Atf4, Atf3, Atf6, Chop, Xbpl, chaperones Hspbl, Hspa5, Hspb7, Dnajc3 and prolyl 4-hydroxylase (P4hb) were increased (Fig. 4A). The activation of the HBP through increase of Gfpt activity was reported to lead to ER-stress trigging the phosphorylation of both PERK (PKR-like endoplasmic reticulum kinase) and eIF2a (eukaryotic initiation factor 2) as well as downstream ATF4 (Activating Transcription Factor 4) translation, identifying the HBP as a modulator of the ISR.

[0107] To determine the relation between IRS and HBP in the vasculature, the inventors treated ShFbnl -VSMCs with DON for 48h. DON decreased IRS signaling through Atf4, Atf6 protein and mRNA expression (Fig. 4B, C). To confirm that excessive HBP activity induces IRS in VSMCs the inventors analyzed Atf4 and Atf6 expression in VSMCs transduced with a lentivector that overexpresses GFPT2 (LV-GFPT2); indicating an activation of ISR (Fig. 4D, E). To assess whether the HBP activity induces ISR in MFS in vivo, the inventors performed a histological analysis of p-pERK, p-eIF2a and Atf4 staining in control and DON-treated FbnlCI039G / +mice (same cohort of mice showed in Fig. 3D) (Fig. 4F). Moreover, the inventors analyzed by immunoblot O-GlcNAc, p-PERK, p-eIF2a and ATF4 protein levels (Fig. 4G) and mRNA levels o Atf4 asx Atf6; and also, Atf4-response gene Tgfb2 (Fig. 4H) in aortic extracts from the same cohort of mice showed in Fig. 3D These data indicate that ISR is activated in MFS-mice and inhibited by DON treatment.

[0108] To characterize the effect of DON treatment at the molecular level, the inventors performed RNA-sequencing on aortas from MFS and control mice in the same cohort of mice showed in Fig. 3D. DON decreased the expression levels of genes related to ER-stress and ISR such as Atf4, Chop, Xbpl, Hsp90bl and Atf4-dependent genes such as Ankrd3, Arhfap23, Hyoul, larsl, Igfp7, Cyclooxygenase-2 (Ptgs2) and Tgfb2 (Fig. 41). Notably, ingenuity analysis predicts a decreased of TGFB-pathway (TGFB upstream regulator, -9,913 activation Z-score, p-value 2,571 O'11) in DON treated mice. DON decreased the expression of conventional MFS- affected genes such as transcripts involved in the TGFp pathway and those encoding ECM- related proteins such as Tgfb3, Cnn2, Serpinel, Thbsl, Pdgfa, Acan, Vcan Sdc4 (Fig. 4J). Thus, reducing HBP fully reverts aortic dilation and the associated aortic wall remodeling such as medial ground substance accumulation herein, CMD; and ISR-signaling in aortas from MFS mice.

[0109] Example 2.5. IRS in aortopathy in MFS

[0110] ISR is a decisive cell response pathway that controls translation and bases a protective response and drive a transcriptional program to maintain and to contribute to organismal fitness. However, when the stress cannot be mitigated, the ISR contributes to a maladaptive stress response such as senescence, cellular dysfunction and cell death. First, the inventors evaluated the ISRIB (an ISR-inhibitor) in MFS in vitro. ISRIB is a small molecule that reverses the consequences of p-eIF2a and it was tested in Alzheimer, traumatic brain injury and aged dementia mouse models. ISRIB incubation in ShFbnl-VSMCs for 48h reduces Atf4, Atf6 mRNA levels (Fig. 5A).

[0111] Then, the inventors evaluated the possible role of ISR in MFS aortic pathology by ISRIB- treatment (ISR-inhibitor) in MFS-mice for 28 days (Fig 5B). ISRIB treatment decreases aortic diameter and blood pressure in Fbnlcl039G / +mice to normal levels (Fig. 5C). These data suggest that maladaptive ISR-response mediates aortic pathology in TAAD.

[0112] Example 2.6. HBP-ISR axis in aortas from human MFS patients

[0113] Next, the inventors assessed whether the contribution of HBP and ISR occurs in MFS-patients. The inventors found that immunostaining of GFPT2 in the medial layer of aortic sections from patients with MFS was significantly increased compared with the aortic sections from organ transplant donors (Fig. 6). Histologic analysis of O-GlcNac and WGA levels confirmed a marked increase of HBP in human aortic MFS-samples (Fig. 6). Of note, immunostainings of pPERK indicate a clear activation of ISR in medial aortic samples from MFS-patients (Fig. 6). Taken together, these data support the idea that HBP-ISR axis might be important mediators of the aortic pathology in human TAAD and warrant evaluation of HBP-ISR inhibitors fort the treatment of TAAD.

[0114] Example 2.7. HBS and IRS are increased in a murine model of atheroslcerotic-abdominal aortic aneurysm (AAA)

[0115] To investigate the contribution of the HBS and IRS in a mouse model of acute atherosclerotic abdominal aortic aortic aneurysms (AAA) and ruptures, ApoE-deficient mice were fed on a western diet for 3 weeks and infused with angiotensin-II for 28 days (ApoE^ AAA mice, Fig. 7A). In challenged mice, hypertension and hypercholesterolemia promote robust aneurysm development and lethal aortic rupture. mRNA levels of HBS markers such as GFPT2 and UAP1 were increased in ApoE^ AAA mice compared with ApoE^ control -mice (Fig. 7B). Moreover, IRS master regulator, ATF4 were increased in AAA-mice compared with ApoE^ control-mice (Fig. 7B). These data indicate that also in AAA pathology HBS and IRS were increased.

[0116] PHASE II 1

[0117] Example 3. Material and methods

[0118] Example 3.1. Animal Procedures

[0119] Marfan mouse model harboring a Fbnlcl041G / +allele (JAX stock #012885). Wild-type littermates ( bril ) served as controls for the Marfan mice. Genotyping of the mice was conducted according to the protocols established by Jackson Laboratories. All mice used in the study were of the C57 / BL6(CRL) background and male. For pharmacological interventions: DON (Sigma- Aldrich), dissolved in saline at a concentration of 5 ng* kg 'day ' . ISRIB-Trans isomer was dissolved in 20% DMSO- 80% PEG400 (MedChemExpress), at a concentration of I mg* kg ' day ' . FR04 was dissolved in 20% DMSO- 40% PEG400 and 50% saline at concentration of lOmg* kg 'day ' . DON, ISRIB and FR054 were infused using subcutaneous osmotic minipumps (Model 2004, Alzet Corp). For control mice, the vehicle (Saline or DMSO / PEG400) was infused by subcutaneous osmotic minipumps. Glucosamine, 5% w / v (Merk), was given in the drinking water. To obtain mouse serum, blood was extracted after sacrifice by CO2 inhalation using the cardiac puncture method, collected and centrifuged for 15 min at 13,000 x g. Animal procedures and experiments complied with all relevant ethical regulations, were accredited by the CNIC and IIS-FJD Ethics Committee and the Madrid regional authorities (ref. PROEX 80 / 16, PROEX 74.7 / 23, PROEX 094.8 / 21), and conformed to EU Directive 2010 / 63 / EU and Recommendation 2007 / 526 / EC regarding the protection of animals used for experimental and other scientific purposes, enacted in Spanish law under Real Decreto 1201 / 2005. Mouse health was daily assessed for signs of discomfort; weight loss; or changes in behavior, mobility and feeding, or drinking habits. Mice were housed in an animal facility under a 12h light / dark cycle at constant temperature and humidity and had access to standard rodent chow and water ad libitum.

[0120] Example 3.2. Ultrasound imaging

[0121] In vivo ultrasound imaging was performed to monitor aortic diameter in isoflurane-anesthetized mice (2% isoflurane). High-frequency ultrasound was conducted using a VEVO 2100 echography device (VisualSonics, Toronto, Canada) with a resolution of 30 microns. Maximal internal aortic diameters were measured at diastole utilizing VEVO 2100 software, version 1.5.0.

[0122] Example 3.3. Cell procedures Primary mouse VSMCs were isolated and cultured as previously described. Briefly, aortic media tissue was digested with a solution of collagenase and elastase (Worthington Biochem) until a single-cell suspension was obtained. All experiments with primary VSMCs were conducted during passages 1-4. Treatments included DON (5nM), ISRIB (IpM) or Puromycin (Ipg / mL for 5 minutes). Lentiviral transduction was performed in VSMCs over 5 hours at a multiplicity of infection = 3. The medium was then replaced with fresh DMEM supplemented with 10% FBS, and cells were cultured for three additional days. The HEK-293T (CRL-1573) and Jurkat (Clone E6-1, TIB- 152) cell lines, required for high-titer lentivirus production and lentivirus titration, respectively, were purchased from ATCC. All cells were tested negative for Mycoplasma contamination.

[0123] Example 3.4. Lentivirus production and transduction

[0124] The lentiviral vectors coding sequences for human-GFPT2-GFP and Mock-GFP were obtained from Origene (RC200519L2); pLVX-ATF4 mScarlet NLS (Addgene plasmid # 115969). Lentivectors expressing shRNA targeting murine Fbnl, as well as control shRNA, were purchased from Sigma-Aldrich. Pseudotyped lentiviruses (VSV-G) were generated by transient calcium phosphate transfection of HEK-293T cells and concentrated from culture supernatant by ultracentrifugation (2 hours at 128,000xg; Ultraclear Tubes; SW28 rotor and Optima L-100 XP Ultracentrifuge; Beckman). The viruses were suspended in cold sterile saline and titrated by transduction of Jurkat cells for 48 hours. Transduction efficiency (GFP-expressing cells and puromycin-resistant cells) and cell death (propidium iodide staining) were quantified by flow cytometry. For in vivo transduction experiments, animals were anesthetized with 2% isoflurane. A virus solution (lOOpl, 5*109viable particles / ml in saline) was inoculated directly into the retro-orbital sinus in C57 / BL6 male mice. Transduction efficiency was analyzed in aortic samples using immunofluorescence for GFP and GFPT2.

[0125] Example 3.5. Real-time and quantitative PCR

[0126] Aortas were extracted following perfusion with 5 ml saline solution, and the adventitia layer was discarded. Tissue frozen in liquid nitrogen was homogenized using a cold mortar and an automatic bead homogenizer (MagNA Lyser, Roche). Total RNA was isolated using Trizol (Life Technologies), with 1 pg of total RNA first digested with DNAse and then reverse- transcribed using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher). Quantitative PCR (qPCR) reactions were performed in triplicate using SYBR master mix (Takara), following the manufacturer’s guidelines. Probe specificity was examined through post- amplification melting-curve analysis, ensuring that only one melting temperature (Tm) peak was produced for each reaction.

[0127] The amount of target mRNA in samples was estimated using the 2-CT relative quantification method, with Gapdh utilized for normalization. Fold ratios were calculated relative to mRNA expression levels from controls.

[0128] Example 3.6. Library preparation and Illumina sequencing

[0129] Aortas were extracted following perfusion with cold saline solution, and the adventitia layer was discarded. RNA from libraries was prepared according to the instructions of the "NEBNext Ultra Directional RNA Library Prep kit for Illumina" (New England Biolabs), following the "Poly(A) mRNA Magnetic Isolation Module" protocol. The input yield of total RNA to start the protocol was >300 ng, quantified using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip kit. The obtained libraries were validated and quantified using an Agilent 2100 Bioanalyzer with a DNA7500 LabChip kit, and an equimolecular pool of libraries was titrated by quantitative PCR using the "Kapa-SYBR FAST qPCR kit for LightCycler480" (Kapa BioSystems) and a reference standard for quantification. Following processing on the Illumina HiSeq 2500 instrument, FastQ files were generated containing nucleotide data and quality scores for each position. RNA-seq reads were mapped to the Mus musculus reference genome, GRCm38.p6, using Hisat2 v2.1.0 software. Reads were then preprocessed with SAMtools vl.7 to transform SAM files into BAM files, which were subsequently sorted. These files served as input for the HTSeq vO.6.1 package, producing a file containing mapped reads per gene for each sample, as defined by the Mus musculus, GRCm38.96 version, gff file. After obtaining HTSeq read counts, the Bioconductor RNA-Seq workflow was followed to detect expression differences of genes using the DESeq2 statistical package. Ingenuity Pathway Analysis (IP A) was employed to identify cellular biological functions, gene clusters, and upstream regulators.

[0130] Example 3.7. Flow Cytometry and immunofluorescence

[0131] For flow cytometry analyses, primary VSMCs (passages between pl-p2) were trypsinized, fixed, permeabilized and sequentially incubated with fluorescein-RL2 antibody (Novus Biologicals), 647-WGA (Invitrogen). Samples were assessed with a FACSCanto II cell analyzer (Becton Dickinson) using DiVA acquisition software and FlowJo VI 0 data analysis software. For immunofluorescence, cells were fixed, permeabilized and incubated with and SMA-cy3 (Sigma) and anti-puromycin647 ThermoFisher antibodies. Example 3.8. Metabolomics and GAGs determination

[0132] 50 pl of serum or plasma samples (stored at -80 °C) and 360 pl of ice-cold methanol were vortexed for 2 min, then shaken in an Eppendorf shaker (Thermomixer R) at 800 rpm, 10 °C for 30 min and centrifuged at 10 °C for 10 min at ~ 14,000 x g. Supernatants (320 pl) were transferred to a clean tube and evaporated to dryness under a stream of nitrogen gas at 40°C. Dried samples were resuspended in 70 pL of acetonitrile-water (80:20, v / v). The residue was reconstituted in 70 pL acetonitrile-water (80:20, v / v). LC-MS / MS analysis was performed using an integrated system composed of Agilent 1260 Infinity II and Ultivo 6465 Triple Quadrupole LC / MS system, which was equipped with an Agilent Jet Stream ESI source and controlled by MassHunter Workstation from Agilent Technologies (Santa Clara, CA, USA). Chromatography separation was performed on an AC QUIT Y UPLC BEH C18 column (1.7 pm, 100 mm x 2.1 mm i.d., Waters) and thermostated at 30 °C. The injection volume for each sample was 3 pL. The mobile phases were (A) water containing 0.1% formic acid (v / v) at pH 3 (adjusted with ammonium hydroxide) and (B) acetonitrile containing 0.01% formic acid (v / v). The analytes were eluted using the following program: 0-4 min, linear gradient 80-40% B; 4-5 min, linear gradient 40-20% B; 5-9 min, isocratic 20% B; 9-10 min, linear gradient 20- 80% B; 10-16 min, isocratic 80% B to re-equilibrate the column. Quantitation by multiple reaction monitoring (MRM) analysis was performed in positive ion mode by monitoring the ion transitions m / z 222.1— >204.0, 222.1— >137.9 and 222.1— >186.0 for GalNac / GLcNAc, and m / z 180.1 — >162.0 and 180.1— >72.1 for Glc. The source and gas parameters for the mass spectrometer were set as follows: ion spray voltage 4.0 kV, gas temperature: 325 °C, gas flow 10 L / min, nebulizer pressure 40 psi, sheath gas temperature: 350 °C, sheath gas flow: 12 L / min.

[0133] For GAGs determination, we utilized the Total Glycosaminoglycans Assay Kit (ab289842, abeam). We employed 10 pl of mice serum or 30 pl of human plasma / serum. Prior to GAGs- Probe incubation, we measured the absorbance of the samples to establish a baseline, which was then subtracted from the absorbance obtained after GAGs-Probe incubation.

[0134] Example 3.9. Immunoblot

[0135] For Western blot (WB) analysis, cells were lysed at 4 °C in RIPA buffer containing protease and phosphatase inhibitors cocktail (Promega / Sigma). Proteins were separated by SDS-PAGE and transferred onto 0.45 pm pore size Immobilon PVDF membrane (Millipore). PVDF membranes were blocked with TBS-T (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% (w / v) milk. Membranes were incubated with primary antibodies diluted 1 / 500 to 1 / 1000 followed by TBS-T washes and incubation with HRP-conjugated secondary antibodies (GE healthcare). The signal was visualized by enhanced chemiluminescence with Luminata Forte Western HRP Substrate (Millipore) and the LightBright3 imaging system. The following antibodies were used: anti-O-GlcNac RL2 monoclonal (Abeam), Anti-GFPT2 (Abeam), anti -U API (NovusBiologicals), anti-pPERK (ThermoFisher), and anti-pEIF2a and ATF4 (Cell signaling), anti Actin-HRP (Abeam) and anti-GAPDH (Abeam).

[0136] Example 3.10. Aortic histology

[0137] After euthanization by CO2 inhalation, mice were perfused with saline. Aortas were then isolated and fixed in 10% formalin overnight at 4 °C. Paraffin cross-sections (5pm) from fixed organs were stained with Alcian blue or Verhoeff elastic-van Gieson (EVG), or they were used for immunohistochemistry or immunofluorescence. Elastic fibers were stained with a modified Verhoeff Van Gieson elastin stain kit (Sigma-Aldrich). Elastic lamina breaks, defined as interruptions in the elastic fibers, were counted in the entire medial layer of three consecutive cross-sections per mouse. For immunostaining, the deparaffinized sections were rehydrated, boiled to retrieve antigens (10 mM citrate buffer, Triton-x 0,05%, pH 6) and blocked for 45 min with 10% goat normal serum, 5% horse serum, Triton-x 0,05% and 2% BSA in PBS. Samples were incubated with the following antibodies for immunohistochemistry or immunofluorescence: monoclonal anti-SMA-Cy3 (1 / 1.000, C6198, Sigma), WGA-647 (1 / 2.000 Invitrogen), polyclonal anti-GFPT2 (1 / 300, Abeam), Chicken anti-GFP (1 / 200, Abeam), monoclonal anti-O-GlcNac (1 / 200, Abeam), polyclonal anti-p-PERK (1 / 300 ThermoFisher) and anti-pEIF2, ATF4 (1 / 50 Cell Signaling). Specificity was determined by substituting primary antibody with unrelated IgG (Santa Cruz). For immunofluorescence, secondary antibodies were Alexa-Fluor-647-conjugated goat anti-rabbit, anti-mouse or antichicken (Invitrogen). Sections were mounted with DAPI in Prolong mounting medium (Invitrogen). Images were acquired at 1024 x 1024 pixels, using a Zeiss-LSM800 and Leica SP5 microscopes with 40 oil-immersion objectives.

[0138] Example 3.11. Human samples

[0139] The study complied with all relevant ethical regulations and was approved by the Clinical Research Ethics Committee of Cantabria (ref. 27 / 2013), the Ethics Committee of Instituto de Salud Carlos III (CEI PI91_2018-v2-Enmienda_2019 and CEI PI 65 2023) and Hospital Fundacion Jimenez Diaz (ref. HBP2023) conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Informed consent was obtained from all human participants or their families. Patient clinical data were retrieved while maintaining anonymity. For histological Ascending aorta samples used as controls were obtained anonymously from multi organ transplant donors. During preparation of the heart for transplantation, excess AsAo tissue was trimmed and harvested for the study. Aortic samples and data from MFS patients included in this study were obtained during elective or emergency surgery for aortic root replacement and provided by the Hospital Universitari Vail d’Hebron Biobank (National Registry of Biobanks B.000018, PT20 / 00107). 8 Control and 8 MFS samples matched for age and sex, were obtained from 4 male and 4 female individuals aged between 27 and 52 years old. Tissues were immediately fixed, kept at room temperature for 48 h, and embedded in paraffin.

[0140] Plasma and serum samples were collected from two cohorts of male and female patients as follows: Cohort-1 included 20 controls (aged between 18-48 years) and 20 MFS-patients (aged between 19-49 years), obtained from Hospital Puerta del Hierro Biobank. Cohort-2 consisted of plasma samples from 16 controls (aged between 28-63 years) and 14 MFS-patients (aged between 20-71 years). Marfan syndrome was confirmed by DNA sequencing, revealing pathological dominant or haploinsufficient mutations in the FBN1 gene. MFS-patients were positive for a history of ascending aortic aneurysms (>4mm). Control patients were defined as negative for Marfan Syndrome and without an evident cardiovascular disorder such as atherosclerosis, heart failure or aneurysm. These samples were obtained and processed in accordance with standard operating procedures.

[0141] Example 3.12. Statistical analysis

[0142] Normality of the data was assessed using the Shapiro-Wilk test, while the equality of variance assumption was evaluated using the F-test. Differences between two groups were analyzed using unpaired Student’s t-test, t-test with Welch's correction for unequal variances, or Mann- Whitney U test where appropriate. Experiments with three or more groups were analyzed by one-way, two-way, repeated-measurements two-way analysis of variance (ANOVA), followed by Newman’s post hoc test as necessary. For statistical analysis of RNA-seq data, p-values were corrected using the false discovery rate (FDR) method (FDR <0.05). GraphPad Prism software 9 was used for all other analyses. Statistical significance was denoted as *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001. Sample sizes were determined empirically based on previous experiences in calculating experimental variability, without the use of statistical methods to predetermine sample size. Outliers were identified and excluded using the ROUT method (Q value = 5%) provided with GraphPad Prism 9 before data analysis. The number of animals used is described in the corresponding figure legends, with all experiments conducted with at least four biological replicates. Experimental groups were balanced in terms of animal age, sex, and weight. Animals were genotyped before experiments and housed together, receiving identical treatment. Sex and age are indicated in the figure legends. Appropriate tests were chosen based on data distribution, with comparable variance between groups in experiments described throughout the manuscript. Randomization was not employed for allocating animals to experimental groups, and investigators were not blinded to group allocation during experiments or outcome assessments.

[0143] Example 4. Results

[0144] Example 4.1 Hexosamine biosynthetic pathway is increased in aortas from a murine model of Marfan Syndrome

[0145] To elucidate the molecular mechanisms underlying aortic medial degeneration, we analyzed our previous transcriptional data using from next-generation sequencing in Fbnlcl041G / +, a MFS-mice model. Remarkably, key features observed in MFS patients, including syndromic phenotype, aortic dilatation, aneurysms, and aortic medial degeneration, are reproduced in FbnlCI04IG / +mice. Aortas from 24-week-old Fbnl+ / +and FbnlCI04IG / +mice were analyzed, showing an intermediate stage of TAAD. Our analysis using Ingenuity Pathway Analysis revealed a significant increase in the expression of genes related to UDP-N-acetyl-D- glucosamine Biosynthesis (also known as the Hexosamine Biosynthetic Pathway, HBP) within the aortas of MFS-mice (p-value= 0.047). Despite being a minor branch of glycolysis, constituting only 2-5% of total glucose metabolism under homeostatic conditions, the HBP plays a crucial role through its final metabolite, UDP-GlcNAc. This serves as substrate for 0 / N- glycosylation, a cornerstone for diverse cellular processes. Notably, UDP-GlcNAc is utilized for post-translational modification of intracellular proteins through O-GlcNAcylation (O- GlcNac) and also contributes to the synthesis and assembly of essential cellular components, including glycolipids, GAGs, PGs, and glycoproteins (Figure 8 A,B)

[0146] Remarkably, in murine MFS-aortas, there was a significant upregulation of mRNA levels of key enzymes involved in the HBP, including the inducible rate-limiting enzyme Glutamine- fructose-6-phosphate transaminase 2 (GJpt2 as well as downstream enzymes such as Uapl (UDP-N-Acetylglucosamine Pyrophosphorylase 1) and Gnpatl (glucosamine-phosphate N- acetyltransferase 1) (Figure 8B). The enzymes O-GlcNAc transferase (Ogf) and O-GlcNAcase (Ogd) (Figure 8B), which mediate the dynamic cycling of O-GlcNac on a wide array of cytosolic, nuclear, and mitochondrial proteins were upregulated (Figure 8A,B). Our analysis also revealed an increase in Golgi enzymes involved in extracellular protein N-glycosylation, including Alpha-1, 3 -Mannosyl-Gly coprotein 2-Beta-N-Acetylglucosaminyltransf erase (Mgat) enzymes; Mgatl, Mgat2, Mgat4a, in MFS-aortas (Figure 8B). Furthermore, the transcripts of genes such as Gne (Glucosamine UDP-N-Acetyl-2-Epimerase / N- Acetylmannosamine Kinase), B3gnt3 (UDP-GlcNAc:BetaGal Beta-1, 3-N-

[0147] Acetylglucosaminyltransf erase 3), and Hasl / Has3 (hyaluronan synthase 1 / 3), involved in the biosynthesis of N-acetylneuraminic acid, poly-N-acetyllactosamine chains, Sialyl LewisX (CD 15s), and hyaluronic acid respectively; and Cd44 (Hyaluronan receptor), were increased in MFS-aortas (Figure 8B). These results were further corroborated by qPCR analysis of aortic extracts from MFS-mice (Figure 8C). Likewise, elevated Gfpt2 mRNA levels were found in aortic-VSMCs from MFS-mice. These findings suggest that the upregulation of HBP and downstream enzymes may contribute to elevated levels of O-GlcNAc, N-glycosylation and GAGs in the aortas of MFS mice.

[0148] Additionally, we conducted an analysis of Gfpt2 mRNA expression in aortas at different ages. Gfpt2 mRNA levels exhibit a significant increase at the very onset of aortopathy, as early as 4 weeks old, in Fbnlcl041G / +mice, even when the aortic phenotype is barely detectable (Figure 8D) MFS-mice aortas show an increase of Gfpt2, Uapl and O-GlcNAc levels by confocal immunostaining (Figure 8E). Immunoblot analysis of aortic samples from MFS-mice revealed elevated protein levels of O-GlcNAc and Gfpt2 (Figure 8F). To assess extracellular GAGs and glycoprotein levels, we used fluorescent Wheat Germ Agglutinin (WGA) that selectively binds to N-acetylglucosamine residues, predominantly present in the extracellular matrix. WGA staining is increased in MFS-mice ascending aortas (Figure 8G).

[0149] In an effort to model MFS in vitro, we utilized lentiviral vectors to silence Fbnl expression in primary murine VSMCs. Consistent with the transcriptomic data obtained from MFS mice, VSMCs with silenced Fbnl (shFbnF) exhibited elevated protein levels of Gfpt2 and Uapl (Figure 8H) and Gjpt2 mRNA levels. Furthermore, shFbn \ -VSMCs demonstrated increased levels of O-GlcNac as determined by immunoblot (Figure 8H) and enhanced WGA and O- GlcNac staining analyzed by flow cytometry analysis. These findings suggest that FBN1 deficiency induces abnormal HBP activity, leading to increased levels of O-GlcNac and WGA-staining in aortic tissue. These changes may contribute to the observed increase in GAGs and PGs observed in aortic medial degeneration in MFS aortas.

[0150] Example 4.2. Hexosamine Biosynthetic Pathway promotes aortic dilation and medial degeneration in wild type- and Marfan Syndrome- mice

[0151] Given that excessive GAGs-glycoprotein accumulation in aortic medial degeneration is a hallmark of TAAD, we hypothesized that increased HBP activity contributes to the production of metabolites required for the PGs and GAGs medial accumulation, therefore, forming the ground-mucoid substance during TAAD development. To investigate this hypothesis, we treated 20-weeks old Fbnl+ / +and FbnlCI04IG / +mice with glucosamine (GlcN) to fuel HBP (Figure 9A). Glucosamine is uptaken by the cells and undergoes transformation to glucosamine-6-phosphate through hexokinases and ultimately converted to UDP-GlcNAc (Figure 9A).

[0152] Treatment with glucosamine in drinking water for 28 days in wild-type mice (Fbril ) led to a significant increase in aortic diameter (Figure 9A-C). Notably, administration of glucosamine to Fbnlcl041G / +mice resulted in a further increase in aortic diameter compared to control Fbnlcl041G / +vawe. (Figure 9B-C). The histological features of aortic medial degeneration were reproduced in Fbnl+'+mice following glucosamine treatment and exacerbated aortic architectural medial abnormalities in Fbnlcl041G / +, such as elastin fiber fragmentation and glycan-matrix accumulation, as evidenced by Alcian Blue staining (Figure 9D-E). Alcian blue is a cationic dye that stains acidic polysaccharides, particularly GAGs. Histological analysis also revealed that glucosamine increased HBP activity by O-GlcNac and WGA confocal analysis in aortic tissue from Fbnl+ / +mice (Figure 9F-G).

[0153] To elucidate the potential role of GFPT2 upregulation in the development of aortic aneurysm and aortic medial degeneration, we transduced primary murine VSMCs with GFPT2 encoding lentiviral vectors (LV-GFPT2). GFPT2 transduction resulted in an increase in O-GlcNac levels (Figure 9H). LV-GFPT2 / / ? vivo transduction in C57BL / 6 wild-type mice (Figure 91), showed that increased GFPT2 expression led to an enlargement of aortic diameter (Figure 9J). To further confirm lentiviral transduction and HBP activity, we performed confocal immunostaining of GFP, GFPT2 and O-GlcNac; WGA respectively (Figure 9K-L). GFPT2 transduction induce an increase in elastin breaks and Alcian Blue staining (Figure 9M), indicating glycan-matrix accumulation.

[0154] These findings together suggest that excessive HBP activity resulting for GFPT2 upregulation contributes to the accumulation of mucoid substance in aortic medial degeneration during TAAD development and is detrimental to aortic homeostasis.

[0155] Example 4.3. Hexosamine Biosynthetic Pathway plays a critical role in the aortopathy of Marfan Syndrome

[0156] To investigate the therapeutic potential of HBP inhibitors in TAAD, we treated FbnlCI04IG / +- or shFbnl- VSMCs with DON (6-diazo-5-oxo-L-norleucine) for 48 hours. DON is a potent HBP inhibitor that blocks the enzymatic activity of GFPT. Treatment with DON efficiently reduced levels of O-GlcNac and WGA in FbnlCI04IG / +- or shFbnl- VSMCs, as evidenced by immunoblot and flow cytometry analysis (Figure 10A). Additionally, DON incubation resulted in decreased mRNA expression of classical MFS genes, such as Tgbl, Fgf2, Osteopontin (SppF) and collagenl (Collal) in shFbnl-VSMCs.

[0157] Next, we evaluated the therapeutic potential of DON in modulating HBP activity during the development of aneurysms in 20 / 22-weeks old MFS mice over a 28-day period was evaluated (Figure 10B). Remarkably, after 28 days of DON-infusion, normalized aortic dilation in FbnlCI04IG / +mice (Figure 10C-D) and restored histological features of aortic degeneration in MFS, including medial thickening, elastic fiber fragmentation, and deposition of GAGs and PGs observed by Alcian blue staining (Figure 10E-F). Furthermore, DON treatment for 28 days also resulted in reduced levels of O-GlcNac and WGA observed by immunoblot and confocal staining in aortic tissue (Figure 10G-H). Notably, DON treatment in FbnlCI04IG / +mice, normalize GAGs and HBP-metabolites in serum, including N-AcetylGlucosamine / N- AcetylGalactosamine (GlcNac / GalcNac), and glucosamine (GlcN) (Figure 101).

[0158] We performed a complementary pharmacological approach to inhibit HBP, by using a specific inhibitor of phosphoacetylglucosamine mutase-3 (PGM3), FR054 (Figure 8A). FR054 treatment has been reported to efficiently reduce both N- and O-glycosylation levels in tumor cells. Treatment with FR054 efficiently reduced levels of O-GlcNAc and WGA in Fbn 1C1O41G / +VSMCs, as evidenced by flow cytometry analysis (Figure HA). Additionally, FR054 incubation led to a reduction in mRNA expression of classical MFS genes, such as Sppl, Fgf2, in aortic VSMCs from Fbnlcl041G / +mice (Figure 11B). Then, the effect of FR054 in modulating HBP activity during MFS-aortopathy development was then evaluated. FR054 or vehicle was infused into 18 / 20-weeks-old MFS mice above a 28-day period (Figure 11C). After 28 days of treatment, FR054 normalized aortic dilation in FbnlCI04IG / +mice (Figure

[0159] HD) and restored the histological features of aortic degeneration in MFS, such as elastic fiber fragmentation and GAGs- and PGs- deposition, as observed by Alcian blue staining (Figure

[0160] HE). Moreover, 28 days of FR054 treatment also reduced O-GlcNAc and WGA levels, as seen through confocal staining in aortic tissue (Figure HF); and normalized GAG levels in serum (Figure HG). FR054 treatment for 28 days, reduced the Col lai and Klf4 mRNA levels in aortas from Fbnlcl041G / +mice. Notably, FR054 treatment in Fbnlcl041G / +mice (Figure 11H).

[0161] These data indicate that HBP inhibition decreases serum GAGs and HBP-metabolites, aortic glycosylation levels, thereby reducing aortic medial degeneration, improving aortic architecture, and restoring homeostasis in a MFS mouse model.

[0162] Example 4.4. Integrated Stress Response is activated by Hexosamine Biosynthetic Pathway in Thoracic Aortic Aneurysm

[0163] The analysis of RNA-seq data from the aortas of 24-week-old MFS-mice revealed the activation of Endoplasmic Reticulum Stress (p-value= 0.032, z-score=1.342). In the aortas of MFS-mice, the expression of genes associated with the Endoplasmic Reticulum-stress profile were increased, including Atf4, Atf3, Atf6, Chop, Xbpl, chaperones such as Hspbl, Hspa5, Hspb7, Dnajc3, and prolyl 4-hydroxylase (P4hb) (Figure 12A). Endoplasmic Reticulum Stress occurs when unfolded or misfolded proteins accumulate in the Endoplasmic Reticulum, disrupting its normal function and leading to cellular perturbations. To cope with Endoplasmic Reticulum-stress, cells activate the Integrated Stress Response (ISR), a complex signaling network. One critical component of the ISR is the phosphorylation of protein kinase PERK (PKR-like endoplasmic reticulum kinase), which phosphorylates the translation initiation factor eIF2a (eukaryotic initiation factor 2a). This phosphorylation event inhibits canonical protein synthesis while promoting the translation of specific mRNAs, including ATF4 (Activating Transcription Factor 4). Through its downstream targets, ATF4 coordinates cellular adaptations aimed at restoring homeostasis and facilitating cell survival in the face of stress. It has been described that the activation of the HBP through increased GFPT activity led to Endoplasmic Reticulum-stress, triggering the phosphorylation of both PERK and eIF2a, as well as downstream ATF4 translation, thereby identifying the HBP as a modulator of ISR. Likewise, DON-treatment reduces to Endoplasmic Reticulum-stress induced by GFPT overexpression or Glucosamine-treatment.

[0164] To determine the potential relationship between ISR and HBP in the vasculature, we treated shFbnl-VSMCs with DON for 48 hours. DON treatment led to a decrease in ISR signaling, evidenced by reduced phosphorylation of eIF2a and decreased protein and mRNA expression of Atf4 and A if 6 . To assess whether the HBP activity induce ISR in MFS in vivo, we performed histological analysis of p-Perk, p-eIF2a and Atf4 staining in control and DON-treated for 28 days Fbnlcl041G / +mice. The results indicated that DON treatment reduces ISR in the aortas of MFS mice. (Figure 12B). Moreover, we analyzed O-GlcNac, p-Perk, p-eIF2a and Atf4 protein levels by immunoblot (Figure 12C) and mRNA levels of Atf4 and Atf6 and Atf4-response gene Tgfb2 (Figure 12D). These data indicate that ISR is activated in MFS-mice and inhibited by DON treatment.

[0165] To characterize the molecular impact of DON treatment, we conducted RNA-sequencing analysis on aortas obtained from Fbnl+ / +and Fbnlcl041G / +mice treated with or without DON for 28 days (n=4). Our results revealed that DON treatment led to decreased expression of genes associated with the ISR, including Atf4 and Atf4-dependent genes such as Chop, Xbpl, Hsp90bl, Ankrd3, Arhfap23, Hyoul, larsl, Igfp7, Cyclooxygenase-2 (Ptgs2), and Tgfb2 (Fig. 5E). Remarkably, Ingenuity Pathway Analysis predicted a reduction in the TGFP pathway activity in DON-treated mice, with a significant decrease in TGFP upstream regulator activation Z- score (p-value= 2.57* 10'11; activation Z-score= -9.913). Furthermore, DON treatment downregulated the expression of conventional MFS-affected genes, including transcripts involved in the TGFP pathway and those encoding extracellular matrix-related proteins such as Tgfb3, Cnn2, Serpinel, Thbsl, Pdgf; and the proteoglycans Acan, Vcan, and Sdc4 (Figure 12E).

[0166] To confirm that excessive HBP activity induces ISR in VSMCs, we analyzed eIF2a phosphorylation and Atf4 and Atf6 protein and mRNA gene expression in VSMCs transduced with a LV-GFPT2. This analysis revealed an activation of ISR. Our in vivo data, revealed that augmenting HBP activity through glucosamine supplementation or LV-GFPT2 transduction in C57BL / 6 wild-type mice for 28 days, leads to ISR signaling. This was evidenced by an increase in p-Perk, p-eIF2a, and Atf4 aortic immunostainings (Figure 12F-G). These findings indicate that excessive protein glycosylation induced by HBP activity triggers ISR signalling in aortas from wild type- and MFS- mice.

[0167] Example 4.5. Integrated Stress Response is involved in vascular smooth muscle cell dysfunction in Marfan syndrome.

[0168] The ISR is a crucial cellular response pathway that regulates canonical protein translation rate and orchestrates a protective response, aiming to maintain cellular homeostasis and contribute to organismal fitness. Initially, we examined Atf4 translation in MFS by transducing primary VSMCs from FbnlCI04IG / +and Fbnl+ / +mice or wildtype- shControl and F / w / -silenced with a lentiviral vector carrying a reporter for Atf4 translation. This lentiviral vector contained a fluorescent protein, Scarlet, fused with the 5' untranslated region of ATF4 (LV-ATF4 Scarlet)37. Both shFbnl-VSMCs and VSMCs from Fbn 1C1041G / +mice demonstrated an increase in Scarlet fluorescence compared to shControl or Fbnl+ / +-VSMCs, indicating enhanced ATF4 translation in Fbnl deficient cells (Figure 13A). Furthermore, treatment of VSMCs- Fbnlcl041G / +with DON for 24 hours resulted in a decrease in Scarlet fluorescence compared to control VSMCs-Fbnlcl041G / +, pointing to the involvement of HBP in ATF4 translation (Figure 13A)

[0169] Next, we explored the role of ISR in MFS. Initially, we assessed the efficacy of ISR inhibitor (ISRIB) in vitro. ISRIB is a small molecule known to reverse the effects of p-eIF2a and has been investigated in various mouse models, including Alzheimer's disease, traumatic brain injury, and dementia. Treatment with ISRIB for 24 hours in shFbnl-VSMCs resulted in reduced levels o Atf4 an A if 6 mRNA. Additionally, ISRIB treatment for 24 hours in VSMCs- F6w7c;cwG / +transduced with LV-ATF4 Scarlet led to decreased Scarlet fluorescence, indicating the involvement of ISR in ATF4 translation in Fbnlcl041G / +-VSMCs (Figure 13A). The ISR decreases canonical translation rate through the phosphorylation of eIF2a. To further investigate the translation rate in MFS, we employed puromycin, an antibiotic that incorporates into nascent proteins, allowing visualization of actively translating ribosomes. After treating FbnlCI04IG / +or Fbnl+ / +VSMCs with puromycin for 5 minutes, we analyzed the puromycin labeled proteins by immunostaining. Fbnlcl041G / +-VSMCs exhibited a significant decrease in puromycylated proteins compared to Fbnl+ / +cells, indicating a lower rate of protein translation in Fbnl mutant cells (Figure 13B). Conversely, treatment with DON and ISRIB for 24 hours in FbnlCI04IG / +-VSMCs increased puromycin labelled proteins (Figure 13B). Flow cytometry analysis reveals that FR054 or ISRIB treatment for 24 hours in FbnlCI04IG / +aortic VSMCs increases puromycin-labeled protein synthesis. Incubation with FR054 for 24 hours decreases Atf4 and Tgfb2 mRNA levels in FbnlCI04IG / +aortic VSMCs. Similarly, as observed in vitro, FR054 infusion in FbnlCI04IG / +mice for 28 days decreases p-Perk, p-Eif2a, and Atf4 aortic immunostaining, indicating a reduction in ISR signaling. Additionally, aortic extracts from FbnlCI04IG / +mice treated with FR054 for 28 days show a decrease in Atf4, Atf6, and Tgfb2 mRNA levels. These findings indicate that HBP induces a reduction in translation rate through the activation of ISR in MFS.

[0170] Subsequently, we evaluated the possible role of ISR in MFS aortic pathology in vivo by infusing 20 / 22-weeks old Fbnlcl041G / +mice with ISRIB for 28 days (Figure 13C). Remarkably, ISRIB-treatment led to a decrease in aortic diameter in FbnlCI04IG / +mice, restoring them to normal levels (Figure 13D-E). Moreover, ISRIB treatment notably improved aortic histology, as evidenced by reduced elastic fiber fragmentation and diminished Alcian blue staining in MFS-mice (Figure 13F-G). Additionally, ISRIB treatment led to a reduction in Atf4 protein levels as observed through immunostaining and immunoblot analysis (Figure 13H-I) along with decreased expression of IRS-induced genes such as Atf4, Atf6 and Tgfb2 (Figure 13J). These data suggest that maladaptive ISR-response mediates aortic pathology in TA AD associated with MFS.

[0171] Example 4.6. Hexosamine Biosynthetic Pathway and Integrated Stress Response axis in aortas from patients with Marfan Syndrome

[0172] Next, we pursued to determine whether the involvement of HBP and ISR extends to patients with MFS. Histologic analysis of O-GlcNac and WGA staining confirmed a significant increase in HBP activity in human aortic MFS samples (Figure 14A,B). Furthermore, in two distinct patient cohorts, MFS patients exhibited elevated serum and plasma levels of GAGs compared to controls (Figure 14C). Through metabolomic analysis, we observed an elevation in plasma HBP metabolites, specifically N-AcetylGlucosamine / N-AcetylGalactosamine (GlcNAc / GalNAc) and glucosamine (GlcN) (Figure 14D). Furthermore, immunostaining of GFPT2 in the medial layer of aortic sections from MFS patients showed a significant increase compared to sections from organ transplant donors (Figure 14E,F). Notably, immunostaining of p-PERK, p-EIF2a, and ATF4 indicated a clear activation of ISR in medial aortic samples from MFS patients (Figure 14E,F). Taken together, these findings support the notion that the HBP-ISR axis may serve as important molecular markers and mediators of aortic pathology in human TAAD. Consequently, the evaluation of HBP-ISR inhibitors for the treatment of TAAD is warranted (Figure 14G).

Claims

CLAIMS1. In vitro method for the diagnosis of aortic aneurysm, which comprises: a) assessing the level of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list consisting of: N-AcetylGlucosamine, N-AcetylGalactosamine or glucosamine, or glycosaminoglycans comprising thereof, in a biological sample obtained from a subject, b) wherein a higher level, as compared with a pre-established threshold value determined in subjects who are not suffering from aortic aneurysm, is an indication that the subject is suffering from aortic aneurysm.

2. In vitro method, according to claim 1, wherein the aortic aneurysm is selected from abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated to atherosclerosis, or thoracic aortic aneurysm, preferably aneurysm associated to Marfan syndrome.

3. In vitro method, according to any of the previous claims, wherein the biological sample is selected from: aortic tissue, serum, plasma or blood.

4. In vitro use of a nucleotide sugar of the hexosamine biosynthetic pathway selected from the list consisting of: N-AcetylGlucosamine, N-AcetylGalactosamine or glucosamine, or glycosaminoglycans comprising thereof, or use of a kit comprising reagents for the detection of a nucleotide sugar selected from the list consisting of: N- AcetylGlucosamine, N-AcetylGalactosamine or glucosamine, or glycosaminoglycans comprising thereof, for the diagnosis of aortic aneurysm.

5. In vitro use, according to claim 4, wherein the aortic aneurysm is selected from abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated to atherosclerosis, or thoracic aortic aneurysm, preferably aneurysm is associated to Marfan syndrome.

6. Inhibitor of an enzyme involved in the hexosamine biosynthetic pathway and / or of an enzyme downstream the hexosamine pathway and / or an enzyme involved in the integrated stress response (ISR), selected from FR054 with CAS No.: 35954-65-5, 6- diazo-5-oxo-norleucine (DON) with CAS No.: 157-03-9 or ISRIB with CAS No.: 1597403-47-8 for use in a method for the prevention and / or treatment of aortic aneurysm.

7. Inhibitor for use, according to claim 6, wherein the aortic aneurysm is selected from abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated toatherosclerosis, or thoracic aortic aneurysm, preferably aneurysm associated to Marfan syndrome.

8. Composition comprising any of the inhibitors of claim 6 and, optionally, pharmaceutically acceptable excipients or carriers for use in the prevention and / or treatment of aortic aneurysm.

9. Composition for use, according to claim 8, wherein the aortic aneurysm is selected abdominal aortic aneurysm, preferably abdominal aortic aneurysm associated to atherosclerosis, or thoracic aortic aneurysm, preferably aneurysm associated to Marfan syndrome.

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