WO2022049320A1 - Nad+ precursors for use in the prevention and/or treatment of hereditary aortic aneurysms - Google Patents

Abstract

The invention relates to a nicotinamide adenine dinucleotide (NAD+) precursor, or a pharmaceutical composition comprising same, for use in the prevention of the development of hereditary aortic aneurysms characterised by the presence of impaired mitochondrial respiration, and/or the treatment thereof. The invention also relates to a nutraceutical composition comprising a nicotinamide adenine dinucleotide (NAD+) precursor for use in preventing the development of hereditary aortic aneurysms characterised by the presence of impaired mitochondrial respiration.

Description

1

PRECURSORS OF NAD ⁺ FOR USE IN THE PREVENTION AND/OR TREATMENT OF HEREDITARY AORTIC ANEURYSMS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a nicotinamide adenine dinucleotide (NAD+) precursor, or to a pharmaceutical composition comprising it, for use in the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology characterized by the presence of impaired mitochondrial respiration. Additionally, the present invention also relates to a nutraceutical composition comprising a nicotinamide adenine dinucleotide (NAD+) precursor, for use in preventing the appearance of an aortic aneurysm of hereditary etiology characterized by the presence of poor mitochondrial respiration. .

BACKGROUND OF THE INVENTION

Aortic aneurysm (AA) is a complex vascular pathology characterized by permanent dilation of the blood vessel wall.

Depending on their etiology, two types of AA can be distinguished: aortic aneurysms caused by a degenerative process, associated with cardiovascular risk factors, and aortic aneurysms associated with hereditary disorders (HAA). The latter are associated with heritable mutations in several genes, including those that code for extracellular matrix proteins (ECM), members of the TGFp signaling pathway, and cytoskeletal actomyosin components of vascular smooth muscle cells (VSMCs).

There are mainly five autosomal dominant disorders currently known that affect the structure of the arterial walls: Marfan syndrome, Loeys-Dietz syndrome, vascular-type Ehlers-Danlos syndrome, Cutis laxa syndrome, and the vascular forms. Family members of non-syndromic Thoracic Aneurysms and Dissections. Marfan syndrome is the most common AAH, it is related to mutations in the extracellular protein fibrillin-1 and its typical cardiovascular symptoms are aortic dilatation and mitral valve dissection and prolapse. Their cardiovascular alterations are accompanied by characteristic musculoskeletal pathologies and lens dislocation.

AAH is a typically asymptomatic process that, therefore, is often undiagnosed and its progression is associated with devastating consequences, such as aortic rupture and sudden death due to uncontrolled bleeding. Unfortunately, there are currently only limited pharmacological treatments to delay the progression of these AHAs, but there is no treatment to reverse them. In fact, the The only 2 options capable of preventing aneurysm rupture are endovascular repair or surgery. For this reason, there is an urgent need to identify the molecular mechanisms involved in AA and, in particular, in hereditary AA to develop pharmacological approaches that not only prevent, but also reverse said aortic aneurysms.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the present invention refers to a precursor of nicotinamide adenine dinucleotide (NAD+), for use in the prevention of the appearance and/or the treatment of an aortic aneurysm of hereditary etiology that is characterized by the presence of a deficient mitochondrial respiration. .

Another aspect of the present invention relates to a pharmaceutical composition comprising a nicotinamide adenine dinucleotide (NAD+) precursor and at least one nutraceutically acceptable excipient, for use in preventing the onset and/or treatment of an aortic aneurysm of etiology inherited characterized by the presence of impaired mitochondrial respiration.

Additionally, the present invention also relates to a nutraceutical composition comprising a nicotinamide adenine dinucleotide (NAD+) precursor and at least one nutraceutically acceptable excipient, for use in preventing the appearance of an aortic aneurysm of hereditary etiology characterized by the presence of impaired mitochondrial respiration.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Decline of mitochondrial function in Marfan syndrome

RNA sequencing analysis of four pb _nicio39G + mice _{with three} raf _Ones Fbn1 ^+/+ from the same litter (20-week-old male mice). (1A) Prediction of the top 10 canonical pathways predicted by Ingenuity Pathway Analysis (IPA) based on differentially regulated genes. Pathways related to metabolism are marked in bold; P<0.01. (1B) Activation or inhibition of major regulators predicted by IPA analysis (corrected deviations>-2 and z-score->4;P<0.05). (1C) Expression of genes encoding mitochondrial complexes (Co) and fatty acid beta-oxidation enzymes, and genes related to mitochondrial function and glycolysis; p<0.05. (1 D) RT-qPCR analysis of Tfam mRNA expression and (1 E) qPCR analysis of relative mtDNA content, in aortic extracts from pb _n i ^c i°39G + and pb _n +/+ q _e 20 mice weeks old. (1F) RT-qPCR analysis of Tfam and PPargd mRNA expression and (1G) qPCR analysis of content 3 relative DNAmt, in VSMCs transduced with shFbnl- and with respect to shControl. (1 H) Oxygen consumption rate (OCR) in shFbnl- and shControl VSMCs under basal respiration conditions (middle) and after addition of complex V inhibitor oligomycin (I) and fluorocarbonyl cyano-phenylhydrazone (FCCP) (II) to measure maximal respiration (right), followed by a combination of rotenone and antimycin A (III). (11) Extracellular lactate levels in supernatants of VSMCs shFbnl- and shControl. Analysis of human ascending aorta samples from Marfan syndrome patients and control donors. (1 J) mtDNA content and (1 K) mRNA expression of genes encoding components of mitochondrial complexes and of genes related to mitochondrial function and glycolysis. (1 L) Immunohistochemistry and quantification. Analysis of primary dermal fibroblasts from 4 Marfan syndrome patients and 4 healthy individuals (control): (1 M and 1 N) OCR on dermal fibroblasts from human samples with Marfan syndrome and controls under basal breathing conditions and after respiration. addition of oligomycin (I), (FCCP) (II) and a combination of rotenone and antimycin A (III); (10) extracellular lactate levels; (1P) qPCR analysis of mtDNA content and PCTq-RT analysis of TFAM and PPARGC1A mRNA expression; (1Q) RT-qPCR analysis of mRNA expression of MT-ND1, SDHA, CYCS, MT-CO1, MY-ATP6 (upper plot), UCP2, HIF1A, and MYC (lower plot). Data from (1 RD) are means ± mean standard error. Statistical significance was evaluated by the t-Student test. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 vs. control mice, Fbn1+/+ mice (D, E), shControl (F, G, H, I) , or healthy donors (JQ).

Figure 2: Fbn1 mRNA levels.

Fbn1 mRNA expression after transduction with shFbnl or shControl.

Figure 3: Conditional ablation of Tfam in VSMCs produces a predisposition to aneurysms and aortic dissections similar to AAH model mice.

(3A) qPCR-RT analysis of Tfam expression (left) and qPCR analysis of mtDNA content (right) in extracts of aortas from SM-Tfam ^+/+ and SM-Tfam'' mice after treatment with tamoxifen (Tmx ) for 28 weeks. (3B) Percentage of survival after treatment with Tmx injections in SM-Tfam ^+/+ and SM-Tfanr" mice, n=20. (3C) Evolution of blood pressure (diastolic BP -right- and systolic BP -left- after treatment with Tmx injections in SM-Tfam ^+/+ and SM-TfamY mice (3D) Evolution of the maximum diameters of the ascending aorta (AsAo -left) and abdominal aorta (AbAo -right) after treatment with Tmx in SM-Tfam ^+/+ and SM-TfamY mice (3E) Experimental design for the assays in Figure 3H: 56 days (8 weeks) 4 after treatment with Tmx injections, mini-infusion pumps of the hypertensive Angiotensin-ll (Angll) were implanted in 6 SM-Tfam ^+/+ mice and 10 SM-Tfarrr' mice. Ultrasound and blood pressure (BP) analysis were performed 6 times (open triangles). (3F) Evolution of systolic (top-left) and diastolic (top-right) blood pressure and of the maximum diameters AsAo (bottom-left) and AbAo (bottom-right) after AngII infusion. (3G) Incidence of aortic aneurysms (left) and percent survival (right) of SM-Tfam ^+/+ and SM-Tfanr' mice with AngII from the same cohort as the 3E mice. (3H) Incidence and location of aortic dissections (left) and intramural hematomas (IMH) (right) in the same cohort of mice shown in 3E. (A, C, D, F) Data with mean ± mean standard error are shown. Statistical significance was analyzed using the t-Student method (A), by log-rank (Mantel-Cox) (B,) or by bidirectional ANOVA (C, D, F).

Figure 4: In vitro ablation of Tfam in VSMCs.

Primary VSMCs from Tfamflx/flx mice were transduced with lentiviral vectors expressing GFP (green fluorescent protein, LV-Mock) or those expressing Cre (LV-Cre) and analyzed 10 days later. (4A) RT-qPCR analysis of relative mRNA expression of Tfam, Mt-Nd1, Mt-Co1 and Slc2a1. (4B) qPCR analysis of relative mtDNA content. (4C) Oxygen consumption rate (OCR) under basal respiration conditions (lower-left) and after the addition of oligomycin (I) and FCCP (II) to measure maximal respiration (lower-right), followed by a combination of rotenone and antimycin A (III). (4D) Normalized extracellular lactate levels. (4E) qPCR-RT evaluation of relative mRNA expression of smooth muscle contractile genes Myh11, Acta2, Cnn1, and Smtn. (4F) RT-qPCR evaluation of relative mRNA expression of VSMC synthetic phenotype genes: Spp1, Nos2, Mmp9, and Mmp2.

Figure 5: Nicotinamide ribonucleoside treatment improves aortic homeostasis in a mouse model of Marfan syndrome

(5A) Experimental design: 16-week-old male control (Fbn1 ^+/+ ) and Marfan Syndrome model (Fbn1 ^c1039G/+ ) mice were treated with nicotinamide ribonucleoside (NR) or vehicle for 28 days (arrows). Ultrasound and blood pressure analysis were performed 5 times (triangles): n=9. (5B) Evolution of the maximum diameter of AsAo (top-left) and AbAo (top-right) and diastolic (bottom-left) and systolic (bottom-right) blood pressure during treatment with NR; n=9. (5C) Quantification of elastic band breaks in AsAo (left) and AbAo (middle) and media thickness 5 aortic (right). (5D) RT-qPCR analysis of Tfam (left) and mt-Co1 (right) mRNA. (5E) Analysis of relative mtDNA content. P<0.05, changes in logarithmic scale. Statistical analysis was evaluated by bidirectional (C) or unidirectional (EG) ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001 for _Fp start _39G /+ _{mice Fbn1} ^+/+ controls # P < 0.05, ####P < 0. for Fbn1 ^c1039G/+ NR mice versus control Fbn1 ^c1039G/+ mice.

Figure 6: Nicotinamide ribonucleoside treatment improves mitochondrial respiration and contractile phenotype in Marfan syndrome (MFS) cells

Primary VSMCs, transduced with shFbnl or shControl, were treated with nicotinamide ribonucleoside (NR) for 5 days. (6A) Oxygen consumption rate (OCR) in shFbnl and shControl VSMCs incubated with or without NR, under basal respiration conditions (lower-left) and after the addition of oligomycin (I) and FCCP (II) to measure the maximal respiration (bottom-right), followed by a combination of rotenone and antimycin A (III). (6B) Extracellular lactate levels. (6C) qPCR analysis of mtDNA content. (6D) RT-qPCR analysis of mRNA expression of Tfam (left), Ppargda (middle), and Mt-Co1 (right). (6E) RT-qPCR analysis of mRNA expression of Mmp9, Mmp2, Spp1, and Col1a1. Effect of NR treatment on dermal fibroblasts from 4 Marfan syndrome patients and 4 healthy donors (control); cells were treated with NR for 5 days. (6F) OCR (left) under basal respiration conditions (middle) after addition of oligomycin (I) and FCCP (II) to measure maximal respiration (right), followed by a combination of rotenone and antimycin A (III) . (6G) Extracellular lactate levels. (6H) qPCR analysis of mtDNA content. (6I) mRNA expression analysis of MTCO1, MT-ND6, TFAM, and HIF1A. (6J) mRNA expression analysis of COL1A1, ACAN, CNN2, and TGFB3. Statistical significance was assessed by one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. ShControl (A-E) or vs. Control (F-J); # P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.0001 against ShFbnl (A-E) or against MFS (F-J).

Figure 7: NR treatment of in vitro models of Familial forms of non-syndromic Thoracic Aortic Aneurysms and Dissections (FTAAD) and Loeys-Dietz syndrome (LDS).

Effect of NR on primary mouse VSMCs overexpressing ACTA2 ^R178H (7A1) or TGFBR2 ^G317W (7A2) as models of FTAAD and LDS, respectively. VSMCs were treated for 5 days with NR. (7A) OCR (left) under basal respiration conditions (middle) and after the addition of oligomycin (I) and FCCP (II) to measure maximal respiration (right), followed by a combination of rotenone and antimycin A (III ). bottom panel 6 shows the extracellular levels of lactate. (7B) qPCR analysis of relative mtDNA content. (7C) RT-qPCR analysis of mRNA expression of Tfam, Mt-Co1 and Ppargda. (7D) RT-qPCR analysis of mRNA expression of Mmp9, Mmp2, Spp1, and Col1a1. Data shown are means ± mean standard error. Statistical significance was assessed by one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control;

# P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.0001 versus NR treatment.

Figure 8: NR treatment in female Marfan syndrome model mice.

(8A) Experimental design: Fbn1 ^+/+ and pbn1 ^c1039G/+ female mice

weeks of age were treated with vehicle or NR for 28 days: n=4 Fbn1 ^+/+ mice n=3 Pbn1 ^c1039G/+ mice t _ra t _ac | _{0S with} vehicle; n=3 pbn1 ^c1039G/+ mice treated with NR. Ultrasound and blood pressure (BP) analysis were performed 5 times (triangles). (8B) Maximum diameters of AsAo (ascending aorta - top/left) and AbAo (abdominal aorta - top/right) and diastolic (bottom/right) and systolic (bottom/left) BP after NR treatment. (8C) Quantification of elastin breaks in the AsAo (left) and aortic medial thickness (right). Statistical significance was assessed by bidirectional (8B) or unidirectional (8C) ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001 for Pbn1 ^c1039G/+ versus pbn1 ^c1039G/+ treated with NR.

DESCRIPTION OF THE INVENTION

Although hereditary aortic aneurysms are diseases that present significant differences in etiology, the present invention shows that the contractile phenotype changes that occur in these hereditary diseases are modulated by a decline in the mitochondrial metabolism of vascular smooth muscle cells (VSMCs). ) and that enhancing mitochondrial metabolism is an appropriate therapeutic approach to restore VSMC homeostasis and prevent the evolution and development of aortic aneurysms in these patients. The present invention describes how the precursors of nicotinamide adenine dinucleotide (NAD+) are capable of increasing the function of mitochondrial respiration, preventing and providing treatment for diseases that occur with poor mitochondrial respiration and, specifically, for the prevention of occurrence and treatment of hereditary aortic aneurysms.

Therefore, the present invention refers to a precursor of nicotinamide adenine dinucleotide (NAD+), for use in the prevention of the appearance and/or the treatment of an aortic aneurysm of hereditary etiology (HAA) characterized by the presence of a poor mitochondrial respiration. 7

The present invention discloses several in vitro and in vivo assays performed with samples from human Marfan syndrome patients, mouse models of Marfan syndrome and VSMC models of other AAH such as Loeys-Dietz syndrome and Familial forms of Dissections. and Non-syndromic Thoracic Aneurysms. These trials show how, in all cases, AAH show a common etiology in which there is a decline in mitochondrial respiration.

The animal model of Marfan syndrome, the pb _nicio39G + mouse reproduces aortic dilatation, aneurysms, and histologic features of median aortic degeneration found in patients with Marfan syndrome. As shown in example 1, relating to the molecular mechanisms underlying the formation of AA of hereditary etiology, transcriptional analyzes of aortas from pb _n icio39G/+ |_ _{os n} ¡ _ve | _is that mtDNA _and Tfam mRNA in pb _n i ^c103 9G + aortas showed a lower than normal mtDNA content. In addition, this model showed a reduction in mitochondrial respiration, accompanied by an increase in extracellular lactate production, an indicator of glycolysis.

On the other hand, to model Marfan syndrome in vitro, the Fbn1 gene was silenced in primary murine VSMCs. The data obtained in vitro confirmed the data obtained with the Marfan model mice. Furthermore, flow-through analysis of oxygen consumption rate (OCR) revealed reduced mitochondrial respiration in Fbnf-silenced cells, accompanied by increased extracellular lactate production, an indicator of glycolysis.

To confirm these observations in human samples, mtDNA and mitochondrial function gene expression were measured in aortas from Marfan syndrome patients. In this regard, the data from Example 1 indicate that aortic aneurysm samples from Marfan syndrome patients showed significantly lower levels of mtDNA and reduced transcription of genes encoding mitochondrial complexes or involving mitochondrial function, further showing a upregulation of genes involved in mitochondrial uncoupling and glycolytic redirection of metabolism. Furthermore, analysis of smooth muscle actin (SMA) confirmed that the downregulation of mitochondrial genes was specific to VSMCs.

The data obtained in example 1 shows that both the aortas of Marfan syndrome mice and the aortas of human patients suffer from a decline in mitochondrial respiration. Furthermore, this decline in mitochondrial respiration implies a loss of oxidative phosphorylation activity (OXPHOS) that is compensated by an increase in the rate of glycolysis. 8

Thus, to investigate whether the redirection of metabolism towards glycolysis in AAH, and in particular in Marfan syndrome, plays a role in the acquisition of a synthetic phenotype (responsible for the synthesis of material that forms the extracellular matrix, such as collagen metalloproteinases and proteoglycans) and, in the progression of aortic disease. Glycolytic metabolism in VSMCs was forced by Tfam depletion in vitro and in vivo (example 2).

For in vitro experiments, aortic VSMCs from Tfam ^flox/flox mice were transduced with lentiviral vectors encoding Cre recombinase (LV-Cre). The results obtained from the analysis of these VSMCs show that mitochondrial respiration controls the contractile properties of these cells, promoting a change in metabolism towards a secretory phenotype.

On the other hand, to analyze the effect of Tfam deletion in VSMCs, in vivo, Tfam ^flox/flox mice were crossed with mice expressing the Cre ^ERT2 fusion protein under the muscle myosin promoter (Myh11-Cre ^ERT2 '). smooth (Myh11) in order to effect an effective abrogation of Tfam expression in aortas of Myh11-Cre ^ERT2 Tfam ^{flox flox} (SM-Tfam~~) mice, but not in Myh11-Cre ^ERT2 Tfam ^m/Wt mice ( SM-Tfam ^+/+ ) used as control. Thus, to assess the response of SM-Tfam mice to a hypertensive test, AngII was infused into SM-Tfam'' and SM-Tfam ^{+ +} mice 56 days after tamoxifen treatment. Histologic analysis of thoracic and abdominal aortic sections showed increased aortic diameter, aortic dissections, intramural hematomas, false lumen formation, and features of medial degeneration, including elastic fiber fragmentation and vascular architecture, demonstrating that decline in Mitochondrial respiration in VSMCs induces lethal aortic aneurysms and dissections in model animal models of Marfan syndrome.

Thus, the present invention shows, both in vitro and in vivo, how in said diseases the development of aortic aneurysms of hereditary etiology is promoted by a dysfunction of mitochondrial respiration specifically in VSMCs.

In a preferred embodiment of the invention, the disease or pathology characterized by the presence of poor mitochondrial respiration is an aortic aneurysm of hereditary aetiology (HAA) and, in particular, said AAH is selected from the group consisting of: syndrome of Marfan, Loeys-Dietz syndrome, Ehlers-Danlos syndrome of vascular type, Cutis laxa syndrome and Familial forms of Non-syndromic Thoracic Aneurysms and Dissections.

In this regard, the present invention shows how the use of NAD+ precursors is an effective therapeutic approach to improve mitochondrial dysfunction in aortic aneurysms. 9 of hereditary aetiology, both preventing the development of these aneurysms and reversing their effects.

Specifically, the examples of the present invention show how nicotinamide ribonucleoside (NR) is one of the different NAD+ precursors and, as it has good bioavailability and pharmacokinetic properties, is capable of rapidly improving mitochondrial metabolism, restoring the transcriptional profile , normalizing aortic functions, restoring the contractile properties of VSMCs and reducing aortic dilation, in vitro and in vivo, in animal models of hereditary aortic aneurysms, preventing lethal aortic dissections. More specifically, examples 3 and 4 of the present invention show, in vivo, how NR prevents, but also reverses, the development of aortic aneurysms in animal models (mice) of Marfan syndrome and, also, in vitro, in VSMCs. models of Marfan syndrome, Loeys-Dietz syndrome and familial forms of non-syndromic thoracic aneurysms and dissections.

Thus, example 3 of the present invention shows how NR improves mitochondrial metabolism and restores contractility in VSMCs that have hereditary aortic aneurysm mutations. On the one hand, treatment with NR increased mitochondrial respiration and decreased lactate production in VSMCs model of Marfan syndrome (shFbnl- VSMCs), restoring the levels found in control VSMCs, increasing the contractility of said cells.

On the other hand, to mimic Familial forms of Thoracic Aneurysms and Dissections and Loeys-Dietz syndrome, lentiviral vectors were used to overexpress ACTA2 ^R179H and TGFBR2 ^G357W , respectively, in VSMCs, which resulted in a reduced rate of VSMC consumption. oxygen content, mtDNA content, and mRNA expression of mitochondrial regulators, while increasing extracellular lactate levels. In this regard, the treatment of these VSMCs, models of Familial forms of Thoracic Aneurysms and Dissections and Loeys-Dietz syndrome, with NR, increased mitochondrial respiration, mtDNA content and mRNA expression of mitochondrial regulators, while decreased extracellular lactate levels. In addition, the use of NR also allowed to increase the contractility in said cells.

These data indicate that in VSMCs affected by genetic aortic aneurysms, there is a loss of oxidative phosphorylation activity towards glycolysis, and that the improvement of the mitochondrial metabolic state in vitro with NR restores their contractility phenotype.

On the other hand, Example 4 shows how NR treatment restores the transcriptional signature (gene expression profile) and contractile properties of aortic smooth muscle. in Marfan syndrome model mice and completely reverses defective aortic wall remodeling, aortic dilatation, and medial degeneration.

Therefore, the present invention relates to a NAD+ precursor for use in preventing the onset and/or treating an aortic aneurysm of hereditary etiology characterized by the presence of "deficient mitochondrial respiration".

Furthermore, the present invention relates to the use of a NAD+ precursor in the preparation of a medicament for the prevention of the appearance and/or the treatment of an aortic aneurysm of hereditary etiology characterized by the presence of deficient mitochondrial respiration.

The present invention also describes a method for the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology that is characterized by the presence of mitochondrial respiration, wherein said method comprises administering to a subject who needs it an effective amount of a NAD+ precursor.

An effective amount is understood, in relation to the therapeutic uses described in the present invention, as that which provides a therapeutic effect without providing unacceptable toxic effects in the patient. The effective amount or dose of the drug depends on the compound and the condition or disease being treated and on, for example, the age, weight and clinical condition of the patient being treated, the form of administration, the patient's medical history, the severity of the disease and the potency of the administered compound.

In a particular embodiment, and for the purposes of the present invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is selected from: niacin (NA) or nicotinic acid; nicotinamide (NAM); nicotinamide riboside (NR); nicotinamide mononucleotide (NMN); nicotinic acid mononucleotide; and nicotinic acid adenine dinucleotide; or combinations thereof.

In a preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is niacin (NA) or nicotinic acid. Niacin is, for the purposes of the present invention, vitamin B3.

In another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide (NAM).

In another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide mononucleotide (NMN).

In another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid mononucleotide. eleven

In another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid adenine dinucleotide.

In a very preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide riboside (NR).

The NAD+ precursors described in the present invention can also be used in pharmaceutical compositions.

Thus, another aspect of the invention relates to a pharmaceutical composition comprising at least one precursor of nicotinamide adenine dinucleotide (NAD+) and at least one pharmaceutically acceptable excipient, for use in preventing the appearance and/or treatment of an aneurysm. aortic of hereditary etiology that is characterized by the presence of poor mitochondrial respiration.

The term "comprises" indicates that it includes a group of certain characteristics (for example, a group of characteristics A, B and C) is interpreted to mean that it includes those characteristics (A, B and C), but that it does not exclude the presence of other characteristics (for example, characteristics D or E), provided that they do not make the claim unworkable. Additionally, the terms "contains", "includes", "has" or "encompasses", and the plural forms thereof, should be taken as synonymous with the term "comprises" for the purposes of the present invention. On the other hand, if the expression "consists of" is used, then no additional features are present in the apparatus/method/product, apart from those following said expression. In this sense, for the purposes of the present invention, the term "comprises" can be replaced by any of the terms "consist of", or "consists essentially of". Accordingly, "comprises" may refer to a group of features A, B, and C, which may additionally include other features, such as E and D, provided that such features do not render the claim unenforceable, but said term "comprises ” also includes the situation where the characteristic group “consists of” or “consists essentially” of A, B and C.

On the other hand, the term "subject" refers, for the purposes of the present invention, to a human or an animal.

The term "pharmaceutically acceptable" means, for purposes of this invention, that which is licensed or licensed by a federal or state government regulatory agency or listed in the European, United States or other generally recognized pharmacopoeia for use in animals. , and more particularly in humans.

One embodiment of the invention relates to the use of a pharmaceutical composition comprising at least one precursor of nicotinamide adenine dinucleotide (NAD+) and at least 12 a pharmaceutically acceptable excipient, in the preparation of a medicament for the prevention of the appearance and/or the treatment of an aortic aneurysm of hereditary etiology that is characterized by the presence of deficient mitochondrial respiration.

Another embodiment refers to a method for the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology that is characterized by the presence of deficient mitochondrial respiration, wherein said method comprises administering to a subject who needs it an amount formulation of a pharmaceutical composition comprising at least one nicotinamide adenine dinucleotide (NAD+) precursor and at least one pharmaceutically acceptable excipient.

The excipient included in the pharmaceutical compositions described in the present invention refers to an inert component such as, but not limited to, co-solvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Any pharmacologically acceptable buffer can be used, such as TRIS or any phosphate buffer.

Said compositions can be included in capsules, tablets, sachets or sachets or any other type of presentation.

To make such compositions, conventional techniques for the preparation of pharmaceutical compositions can be used. For example, the compound of interest may be mixed with a carrier or diluted in a carrier or contained in a carrier in the form of an ampoule, capsule, tablet, sachet, sachet, or other container. When the carrier serves as a solvent, it may be solid, semi-solid or liquid and act as a carrier or medium for said active compound. The compound of interest can be adsorbed on a solid granular medium. Some examples of suitable carriers are water, saline solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrins, amylose, magnesium stearate, talc, gelatin, agar, pectin. , acacia, stearic acid, cellulose alkyl ethers, silicon acid, fatty acids, fatty acid amines, fatty acid mono- and diglycerides, pentaerythrol fatty esters, polyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Also, the vehicle or support can include sustained release materials known in the art, such as glyceryl monostearate or di-sterate alone or mixed with a wax. The formulations may also include wetting agents, emulsifiers, suspending agents, preservatives, sweeteners, or flavoring agents. The compositions may be formulated to provide rapid, sustained, or delayed release of the active agent after it is administered to the patient using methods known in the art. 13

The pharmaceutical compositions can be sterilized and mixed, if desired, with additional agents, emulsifiers, salt to influence osmotic pressure, buffers and/or coloring substances that do not react adversely with the active compounds.

One embodiment relates to the mode of administration, which can be any mode that effectively transports the compound of interest to the desired site of action, such as oral, rectal, or parenteral.

For oral administration, both solid and liquid dosage forms can be prepared. To prepare solid compositions as tablets, the compound of interest is mixed into a formulation with other conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, starch, lactose, acacia, methylcellulose, and functionally similar materials such as pharmaceutical carriers and diluents.

Capsules can be prepared by mixing the compound of interest with a pharmaceutically inert solvent and filling the mixture into a hard gelatin of appropriate size. Soft capsules are prepared with machines for encapsulating suspensions of the compound of interest with an acceptable vegetable oil, light paraffin or inert oil. Liquid dosage forms such as syrups, elixirs and suspensions can also be prepared. The water soluble forms can be dissolved in an aqueous vehicle together with sugar, flavors and preservatives to form a syrup. An elixir is prepared using a hydroalcoholic vehicle (eg ethanol) with suitable sweeteners such as sugar or saccharin, together with aromatic flavoring agents. Suspensions can be prepared with an aqueous vehicle and the aid of a suspending agent such as acacia, tragacanth, methylcellulose, and the like.

For parenteral application, the use of injectable solutions or suspensions for intradermal, intramuscular, intravascular and subcutaneous use are obvious to those skilled in the art.

In addition to the compound of interest, the compositions may include other non-toxic pharmaceutically acceptable diluents and excipients, including vehicles commonly used in pharmaceutical compositions commonly used in humans or animals. The diluent is selected so as not to affect the biological activity of the composition.

Examples of diluents used especially in injectable formulations are organic and inorganic saline solutions, Ringer's solution, dextrose solution and Hank's solution. In addition, the compositions may include additives such as other excipients, adjuvant agents, non-therapeutic and non-immunogenic stabilizers, and the like.

Examples of excipients that may be included in the formulation include, but are not limited to 14 co-solvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Any physiologically acceptable buffer can be used, such as Tris or phosphate buffers. Effective amounts of diluents or additives or excipients are those that are effective to obtain a pharmaceutically acceptable formulation in terms of solubility and biological activity.

Another embodiment relates to the dosage regimen. The term "unit dose" refers to physically discrete units suitable as unitary doses for a subject where each unit contains a predetermined quantity of active material calculated to produce the appropriate therapeutic effect in association with the appropriate diluent, carrier or vehicle.

A preferred embodiment disclosed herein relates to the route of administration, which may be any route that efficiently transports the compound disclosed hereinabove, to the appropriate or desired site of action, such as oral. , nasal, topical, pulmonary, transdermal, or parenteral, eg, rectal, subcutaneous.

The NAD+ precursors, or the pharmaceutical compositions comprising them, described in the present invention, can be used before, in combination with, or after another treatment or therapy that is useful in the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology characterized by the presence of impaired mitochondrial respiration. In one embodiment of the invention said treatment or therapy is a hypotensive or an inhibitor of the Angiotensin pathway. Preferably the hypotensive is a beta-blocker. Non-limiting examples of beta-blockers include, but are not limited to Atenolol, Acebutolol, Betaxolol, Bisoprolol, Esmolol, Nebivol, among others. Also preferably, the Angiotensin pathway inhibitor can be selected from Losarian, Olmesartan, Valsarian or Irbesartan, among others.

Furthermore, the NAD+ precursors, or pharmaceutical compositions comprising them, described in the present invention, can be used before, in combination with, or after another therapeutic component or additional active compound. Said additional therapeutic component or active compound provides additive or synergistic biological activities. For the purposes of the present description, the terms "active compound" or "therapeutic component" should be taken synonymously and mean a chemical or biological entity that exerts therapeutic effects when administered to humans or animals.

The NAD+ precursors described in the present invention can also be used as nutraceuticals or in nutraceutical compositions. This is the case, for example, of vitamin B3 and its derivatives. fifteen

Thus, another aspect of the invention relates to a nutraceutical composition comprising at least one nicotinamide adenine dinucleotide (NAD+) precursor and at least one nutraceutically acceptable excipient, for use in preventing the appearance of an aortic aneurysm of hereditary etiology that It is characterized by the presence of impaired mitochondrial respiration.

Additionally, the present invention also relates to a method of preventing the appearance of an aortic aneurysm of hereditary etiology characterized by the presence of poor mitochondrial respiration, wherein said method comprises administering to a subject in need an effective amount of a nutraceutical composition comprising at least one nicotinamide adenine dinucleotide (NAD+) precursor and at least one nutraceutical acceptable excipient.

An effective amount is understood, in relation to the nutraceutical uses described in the present invention, as that which provides a beneficial effect for the health of the subject who ingests it, especially in the prevention of the appearance of diseases, without providing adverse effects. unacceptable toxicants in the subject.

The term "nutraceutical acceptable" refers, for the purposes of the present invention, to everything that is suitable for use in nutraceutical products.

For the purposes of the present invention, the term "nutraceutical" or "nutraceutical composition" refers to a dietary supplement, to be taken by itself, or in combination with other foods, and that produces a beneficial effect on the health of the subject that consumes it. eat, especially in disease prevention.

For the purposes of the present invention, a nutraceutical composition is understood as a food composition, to be ingested separately or with food, which has a medicinal effect on human health.

Specifically, the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is selected from: niacin (NA) or nicotinic acid; nicotinamide (NAM); nicotinamide riboside (NR); nicotinamide mononucleotide (NMN); nicotinic acid mononucleotide; and nicotinic acid adenine dinucleotide; or combinations thereof.

In a preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is niacin (NA) or nicotinic acid. Niacin is, for the purposes of the present invention, vitamin B3.

In another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide (NAM). 16

In yet another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide mononucleotide (NMN).

In yet another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid mononucleotide.

In yet another preferred embodiment of the invention, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid adenine dinucleotide.

In a particularly preferred embodiment, the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide ribonucleoside (NR).

In another preferred embodiment of the nutraceutical composition for use according to the invention, the nutraceutical composition is incorporated into a food preparation.

The nutraceutical composition for use according to the invention can also be included in a variety of food preparations, for example, products derived from milk, yogurt, curd, cheese (for example, fresh, cream, processed, soft and hard cheese), fermented milk , milk powder, a fermented milk-based product, an ice cream, a fermented cereal-based product, milk-based powder, beverages, and a pet food.

The term "food preparations" is used here in its broadest sense, including any type of product, in any form of presentation, that can be ingested by an animal, but excluding pharmaceutical and veterinary products. Examples of other food preparations are meat products (for example pâtés, frankfurters and salami or meat spreads), chocolate spreads, fillings (for example truffle, cream) and glazes, chocolate, confectionery (for example caramel, fondant or toffee), bakery products (cakes, pastries), sauces and soups, fruit juices and coffee whiteners. Particularly interesting food preparations include food supplements and infant formulas.

The nutraceutical composition for use according to the invention could also be used as an ingredient in other food products. Accordingly, in another aspect of the invention, food preparations containing the composition of the invention together with appropriate amounts of edible ingredients are provided. Preferably, the nutraceutical composition for use according to the invention is a food supplement. For the purposes of the present invention, the term "food supplement" refers to that fraction of food that is used to complete human or animal nutrition. If the nutraceutical composition for use according to the invention is used as a food supplement, it can be administered as such, or it can be mixed with a suitable drinking liquid, such as water, yoghurt, milk or fruit juice, or it can be mixed with solid or liquid foods. . In this In this context, the food supplement may be in the form of tablets, pills, capsules, granules, powders, suspensions, sachets, lozenges, candies, bars, syrups and corresponding forms of administration, usually in unit dosage form.

EXAMPLES

The examples described below are for illustrative purposes and are not intended to limit the scope of the present invention.

The materials and methods used in the examples described herein are detailed below.

Materials and methods

Mice and procedures used with animals

The mouse animal model with the Fbn 1 ^C1O39G/+ allele was previously described by Habashi, JP, et al (Science, 2006. 312(5770): p. 117-21). For specific ablation of Tfam in smooth muscle, Tfam ^flox/flox mice were bred to mice carrying the MyH 11 ^{-Cre ERT2} allele expressing a tamoxifen-inducible Cre recombinase under smooth muscle heavy chain myosin promoter regulatory sequences ( Myh11). Wild-type littermates were used as controls for Marfan mice and Tfam ^vvt/vvt MyH11-Cre ^ERT2 as control for Tfam ^flox/flox MyH11-Cre ^ERT2 . For conditional removal of Tfam in smooth muscle, Myh11-Cre ^ERT2 TFAM ^m/wt and Myh11-Cre ^ERT2 Tfam ^{flox/flox mice aged} 3-4 weeks received injections (ip) of 1 mg tamoxifen (Sigma Aldrich) for 5 consecutive days . AngII was dissolved in saline at a concentration of 1 pg Kg-1 min-1 using subcutaneous osmotic minipumps (Model 2004, Alzet Corp). Nicotinamide ribonucleoside (Novalix) was administered intraperitoneally at a concentration of 1000 mg/Kg in saline medium on alternate days. The mice were housed in pathogen-free animal facilities of the Carlos III National Cardiovascular Research Center (CNIC) and the Severo Ochoa Molecular Biology Center (CBMSO) following the animal care standards of these institutions. The procedures with animals were approved by the Ethics Committee of the CNIC and the CBMSO-Universidad Autónoma de Madrid (UAM) and by the Community of Madrid (Rf. PROEX 283/16) in accordance with European Directive 2010/63EU and the 2007 Recommendation /526/EC in relation to the protection of animals used for experimentation and other scientific tasks, in force through Royal Decree 1201/2005. 18

Determination of blood pressure and capture of in vivo images.

Arterial blood pressure (BP) was measured in the tail using the BP-2000 Blood Pressure Analysis System (Visitech Systems, Apex, NC, USA). Mice were trained to measure PS every day for 5 consecutive days. After the training period, PS was measured one day before treatment to determine baseline PS in each cohort of mice. 15 consecutive measurements of systolic and diastolic PS were taken and the last 10 shots of each mouse were recorded and the mean value was obtained. For in vivo ultrasound imaging, aortic diameter in isoflurane (2%) anesthetized mice was monitored with a VEVO 2100 high-frequency ultrasound scanner (VisualSonics, Toronto, Canada) at 30 micron resolution. Maximum aortic internal diameters were measured in diastole using VEVO 2100 software, version 1.5.0.

Cell procedures.

The isolation and culture of primary mouse vascular smooth muscle cells (VSMC) was described by Esteban, V., et al. (J Exp Med, 2011. 208(10): p. 2125-39). The tissue was digested with a collagenase and elastase solution until a single cell suspension was obtained. All experiments with primary VSMCs were carried out during passages 2-7. Lentiviral transduction was carried out in VSMCs for 5 hr with a multiplicity of infection = 3. The medium was then replaced with fresh DMEM supplemented with 10% FBS and the cells were cultured for 7 days, treated with NR for a further 3 days. and in the absence of serum for 16 hr. HEK-293T (CRL-1573) and Jurkat (Clone E6-1, TIB-152) cell lines, required for the production of high titers of lentiviruses and for lentivirus titer respectively, were purchased from ATTC. Purchased from Coriell Cell Repositories: 4 apparently healthy control males (GM00024, GN01717, GM03652, GM23963) and 4 mismatched males and fibroblasts from Marfan syndrome patients with aortic dissection, two with FBN1 point mutations (GM21499, GM21946) and two with haploinsufficient mutations-FB/VI (GM21983, GM21978). The experiments were carried out for passages 5-10. All cells were mycoplasma negative. For immunostaining, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS for 10 minutes. Samples were incubated for 15 minutes with 1/500 647-Phalloidine (Millipore) and DAPI. Images were taken with a Zeiss-LSM-800 microscope with an oil immersion objective of

19

Lentivirus production and infection.

The Cre and GFP coding sequence was obtained by PCR amplification and cloned into the lentiviral vector pHRSIN (Oller et al. 2015 Mol Cell Biol 2015 3409-22). Lentiviruses expressing murine Fbn1 targeting shRNA and control shRNA were purchased from Sigma Aldrich. Lentiviruses expressing FCTA2 ^R179H and TGFBR2 ^G317W mutations and controls were supplied by Mark Lindsay. Pseudotyped lentiviruses were produced by transient transfection of HEK-293T cells with calcium phosphate and concentrated culture supernatant by ultracentrifugation (2 hr at 128,000xg; Ultraclear Tubes; SW28 rotor and Optima L-100 XP Ultracentrifuge; Beckman). Viruses were suspended in cold sterile PBS and titrated by Jurkat cell transduction for 48 hr. Transduction efficiency (GFP-expressing cells and Puromycin-resistant cells) and cell death (propidium iodide staining) were quantified by flow cytometry (Oller et al., Nat Med, 2017. 23(2): p. 200-212).

Extracellular flux analysis and metabolic assays.

Oxygen consumption rate (OCR) was measured with an XF-96 Extracellular Flux Analyzers (Seahorse Bioscience) in 25,000 mouse aortic VSMCs or in 50,000 human fibroblasts seeded in unbuffered DMEM medium containing 25 mM glucose and CaCh 1 mM. Three measurements were taken under basal conditions and with the addition of oligomycin (1 mM), fluorocarbonyl cyano-phenylhydrazone (FCCP, 1.5 mM) and rotenone (100 nM) + antimycin A (1 mM). Extracellular lactate was determined with the Accutrend ® Plus system (Roche). 20 pL of conditioned medium were analyzed after 48 hours of activation. Lactate measurements were normalized to cell protein extracts.

Real-time and quantitative PCR.

The aortas were removed after perfusion with 5 ml of saline, and the adventitial layer was discarded. Frozen tissue was homogenized with liquid nitrogen using cold binder and an automatic microsphere homogenizer (MagNA Lyzer, Roche). Total RNA was isolated with TRIzol (Life Technologies). Total RNA (1 pg) was first digested with DNase and reverse transcription performed with the 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 with TRIzol, respectively, according to the manufacturer's instructions. DNA was amplified using primers specific for cytochrome c oxidase subunit 1 (mt-Co1) and mitochondrial 16S rRNA, and normalized with nuclear B2M and H2K control genes. Quantitative real-time PCRq was performed with the primers from Tables 1A (mouse primers) and 1B (human primers): Table 1A: Mouse Primers

twenty-one

Table 1B: Human Primers

The qPCR reactions were carried out in triplicate with a SYBR master mix (Promega), according to the manufacturer's instructions. To examine the specificity of the probe, post-amplification melting-curve analyzes were performed. For each reaction only one melting temperature (Tm) was taken. The amount of target mRNA in the mixtures was estimated with the 1-CT relative quantification method, using B2M, YWHAZ and PP1A for normalization. Duplication ratios were calculated based on control mRNA expression levels.

immunoblot

For immunoblot or Western blot analysis, cells were lysed at 4°C in RIPA buffer containing protease and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and transferred to an Immobilion PVDF membrane (Millipore) with a pore size of 0.45 pm. PVDF membranes were blocked with TBS-T (50 pM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% (w/v) milk. Membranes were incubated with primary antibodies at a dilution of 1/500 to 1/1000 followed by washes with TBS-T 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-TFAM (Proteintec), Anti-MT-CO1 (Millipore), anti -VDAC (Abeam), anti-a-tubulin (Cell Signaling).

Library preparation and Illumina sequencing

The aortas were removed after perfusion with a cold saline solution and the adventitial layer was discarded. Frozen tissue was homogenized and total RNA isolated with Trizol (Roche). RNA from libraries was prepared according to the instructions of the “NEBNext Ultra Directional RNA Library Prep kit for Illumina” kit (New England Biolabs), following the 22 “Poly(A) mRNA Magnetic Isolation Module” protocol. Total RNA yield at the beginning of the protocol was >300 ng quantified by an Agilent 2100 Bioanalyzer using the RNA 6000 nano LabChip kit. The obtained libraries were validated and quantified with an Agilent 2100 Bioanalyzer using a DNA7500 LabChip kit and an equimolar set of libraries was titrated by quantitative PCR using the "Kapa-SYBR FAST qPCR kit forLightCycler480" (Kapa BioSystems) and a reference standard for quantification. The library pool was denatured before being seeded at 2.2 pM density in a flow cell, where clusters were formed and sequenced using a NextSeq™ 500 High Output Kit, on a NextSeq500 sequencer in a single 1x75 read. . Approximately 15 million reads were obtained after filtering for each sample. Fastq files were aligned against an Mm version genome using a STAR aligner (Base Space Sequence Hub, Illumina) and expression profiles were determined using CuffDiff2. Ingenuity Pathway Analysis (IPA) was used to identify cell biological functions, gene clusters and regulators.

aortic histology.

After euthanizing the mice by CO 2 inhalation, the aortas were perfused with saline. Aortas were then isolated and fixed in 10% formalin overnight at 4°C. Paraffin sections (5 pm) of fixed organs were stained with Masson-Trichrome, alcian blue, or Verhoeff elastic-van Gieson (EVG), or used for immunohistochemistry or immunofluorescence. Elastic fibers were stained with a modified Verhoeff Van Gieson elastin staining kit (Sigma Aldrich). Elastic lamina tears, defined as breaks in elastic fibers, were counted for the entire medial layer of three consecutive cross-sections in each mouse, using 4-16 mice in each experiment, and the mean number of tears was calculated. For immunostaining, deparaffinized sections were rehydrated, boiled to recover antigens (10 mM citrate buffer, 0.05% Thton-x, pH=6) and blocked for 45 minutes in PBS with 10% normal goat serum, 5% horse, 0.05% Thton-x and 2% BSA. The samples were incubated for immunohistochemistry or immunofluorescence with the following antibodies: anti-SMA (1/500, C6198, Sigma), polyclonal anti-TFAM (1/300, Prointech), polyclonal anti-MT-ND1 (1/300, Proteintech), monoclonal anti-Mt-CO1 (1/300 Invitrogene), polyclonal anti-HIF1A and anti-MYC (1/500 Novus biologicals). Specificity was determined by replacement of primary antibodies with unrelated IgG (Santa Cruz). For immunohistochemistry, endogenous peroxidase and biotin were blocked with 1% H2O2-methanol for 10 minutes and a biotin blocking kit (Vector Laboratories), respectively. Color developed at the same time in all 23 samples with DAB (Vector Laboratories) and sections were counterstained with hematoxylin and mounted in DPX (fluka). For immunofluorescence, Alexa-Fluor-546 conjugated goat anti-rabbit and Alexa-Fluor-647 conjugated goat anti-rabbit secondary antibodies (BD Pharmigen) were used. For F-actin determination, fixed aortas were embedded in OCT (Tissue-Tek Sakura), 5 pm cross-sections were incubated for 30 minutes with 1:1000 Phalloidine-657 (Millipore), and after 10 minutes of incubation with 0.3% Triton X-100 in PBS. Sections were mounted with DAPI in Citifluor AF4 mounting medium (Aname). Images were taken at 1024 x 1024 pixels and 8 bits using a Zeiss-LSM800 microscope with 40x oil immersion objectives.

gelatin zymography

Cell culture supernatants were prepared as described by Oller, J., et al. (Nat Med, 2017. 23(2): p. 200-212). Extracts (15 pg) were fractionated under non-reducing conditions on 10% SDS-polyacylamide gel containing 0.1% gelatin. The gels were washed three times with 2.5% Triton X-100 for 2 hours at room temperature, incubated overnight at 37 °C in 50 mM Tris-HCI pH= 7.5, 10 mM CaCh and 200 mM NaCl, and stained with Coomasie blue. Areas of MMP or gelatinolytic activity were visualized as transparent bands. The images were analyzed with Quantity One software (Bio-Rad).

human samples

The study was approved by the ethics and clinical research committee of the Carlos III Health Institute and the University of Cantabria (B2017/BMD-3676 AORTASANA-CM, ref. 27/2013; respectively). Control aortas were obtained anonymously from transplant organ donors after written consent was obtained from the families. During preparation of the heart for transplantation, excess ascending aortic tissue was collected for study. Samples were obtained from patients during elective or emergency aortic root surgery for aortic aneurysm dissection. The patient's clinical data was collected while maintaining his anonymity. Tissues were immediately fixed, stored at room temperature for 48 h, and embedded in paraffin. DNA and RNA extraction from the paraffin-embedded sections was carried out with the All Prep DNA/RNA FFPE kit (Quiagen).

Statistical analysis

GraphPad Prism 6.01 software was used for analysis. Differences were analyzed by unidirectional, bidirectional, or repeated measures with bidirectional variance analysis (ANOVA) and Newmann's post hoc test (experiments with groups > 3), appropriately. For the survival curves, we analyzed the 24 differences with a log-rank test (Mantel-Cox). Statistical significance was assigned to *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. The sample size was chosen empirically based on previous experience in calculating experimental variability; a statistical method was not used to predetermine the sample size. Outliers were identified and excluded using GraphPad Prism software. The number of animals used is described in the corresponding figure legends. All experiments were carried out with at least 3 biological replicates. The experimental groups were balanced according to animal age, gender and weight. The animals were genotyped before the experiments and all of them were confined together and treated in the same way. Appropriate tests were chosen according to the distribution of the data. The variance was comparable between groups in the experiments described herein. For the rest of the experiments, no randomization was used to assign animals to experimental groups.

Example 1: Metabolic changes in Marfan syndrome.

The pp _n icio39G + mouse animal model reproduces aortic dilatation, aneurysms, and histologic features of medial layer aortic degeneration found in patients with Marfan syndrome. To identify the underlying molecular mechanisms of the formation of AA of hereditary etiology, transcriptional analyzes of aortas from pp _nitio39G /+ mice were carried out thanks to Ingenuity Pathway Analysis (IPA), it was observed that 6 of the 10 most altered canonical mechanisms were related to metabolism, including oxidative phosphorylation, mitochondrial dysfunction, fatty acid oxidation (FAO), and the tricarboxylic acid (TCA) cycle, as shown in Figure 1A. The IPA prediction of important regulators described typical regulators found in Marfan syndrome (MFS) such as inducible nitric oxide synthase (Nos2) and growth factor beta 1 (Tgfbl). This analysis pointed to an increase in the activity of the glycolytic regulator Hypoxia Inducible Factor (Hifla) and a reduction in the activity of regulators of biogenesis and mitochondrial function, including Tfam (Figure 1B). The pp _n i ^c1 °39G + aortas showed reduced expression of all subunits of mitochondrial complexes, including genes encoding the nucleus and mitochondhas, as well as genes related to FAO and mitochondrial biogenesis and function (Tfam, Ppara, Pparg Ppargda, Ppargdb, and Sod2). In contrast, genes involved in mitochondrial uncoupling (Ucp2) and glycolytic redirection of metabolism (Hifla, Myc) were upregulated (Figure 1C). RTq-PCR analysis confirmed the reduced expression of Tfam (Figure 1D). _Tfam control mtDNA levels and relative mtDNA content analysis in pp start _39G + _aortas showed lower than normal mtDNA content (Figure 1E). 25

To model Marfan syndrome in vitro, Fbn1 was silenced in lentiviral vectors in primary murine VSMCs (Figure 2). The shFbnl -CML\/s had significantly reduced expression of Tfam and Ppargda, confirming the data obtained with the Marfan model mice (Figure 1F), which was correlated with a reduction in mtDNA content (Figure 1G). . Flow analysis of the oxygen consumption rate (OCR), index of mitochondrial oxidative phosphorylation (OXPHOS), revealed reduced mitochondrial respiration in Fbn1-silenced cells (Figure 1H). This was accompanied by an increase in extracellular lactate production, an indicator of glycolysis (FIG. 11).

To confirm these observations in human samples, mtDNA and mitochondrial function gene expression were measured in aortas from Marfan syndrome patients. Compared with control samples from healthy subjects, aortic aneurysm samples from Marfan syndrome patients showed significantly lower levels of mtDNA (Figure 1J) and reduced transcription of genes encoding mitochondrial complexes (MT-ND1, SDHA, SDHB, CYCS, MTCO1, and MT-ATP6) or that involve mitochondrial function (TFAM, PPARA, PPARG, and PPARG1A). Furthermore, the aneurysm samples showed an upregulation of genes involved in mitochondrial uncoupling and glycolytic redirection of metabolism (Figure 1K). Histological analysis of the mitochondrial proteins MT-ND1 , SDHA, and TFAM and of the genes related to glycolysis HIF1A and MYC (figure 1L). Smooth muscle actin (SMA) co-immunostaining confirmed that the downregulation of mitochondrial genes was specific to VSMCs. In addition, in vitro analysis of mitochondrial respiratory capacity in primary dermal fibroblast cultures from individuals with Marfan syndrome and from four healthy donors showed the relationship between Marfan syndrome and reduced OCR, elevated lactate production, and lower mtDNA content. (Figures 1M, 1N and 1P). In line with the findings in human Marfan syndrome aortas, primary Marfan syndrome dermal fibroblasts showed low mRNA content of MTND1, SDHA, MT-CO1, MT-AP6, TFAM, and PRARGC1A, and high expression of UCP2, HIF1A and MYC (Fig. 1Q). These data suggest that the aortas of Marfan syndrome mice and human patients suffer from a decline in mitochondrial respiration and a redirection of metabolism toward glycolysis.

Example 2: Glycolytic metabolism in VSMCs causes aortic aneurysms similar to those found in Marfan syndrome

To investigate whether the redirection of metabolism towards glycolysis in Marfan syndrome plays a role in the acquisition of a synthetic phenotype and the progression of aortic disease, the conversion of VSMCs to glycolytic cells was forced by depletion of 26

Tfam in vitro and in vivo. For in vitro experiments, aortic VSMCs from Tfam ^flox/flox mice were transduced with lentiviral vectors encoding Cre recombinase (LV-Cre). Tfam deletion was confirmed by analyzing Tfam mRNA levels and protein levels (Fig. 4A and B) against a control that did not encode Cre recombinase (LV-Mock). Consistent with its regulatory role in mtDNA levels, deletion of Tfam in VSMCs resulted in reduced mtDNA content, reduced expression of mtDNA-encoded genes Mt-Co1 and Mt-Nd1, reduced OCR, and increased lactate production (Figures 4A-D). Drastic decreases in mRNA expression of genes involved in contractility, such as smooth muscle heavy chain myosin (Myh1), smooth muscle actin (Acta2), transgelin (Tglrí), calponin (Cnn), and smoothelin ( Smtri) (fig. 4E). Furthermore, this was accompanied by mRNA expression of genes related to the secretion phenotype, such as osteopoietinal (Spp), metalloproteinase-9 (Mmp9), and inducible nitric oxide synthase (Nos2) (Fig. 4F). The increased expression of Mmp9 was accompanied by a marked increase in Mmp9 enzymatic activity in cell supernatants (FIG. 4F). These data suggest that mitochondrial respiration controls the contractile properties of VSMCs, while glycolytic conditions move these cells toward a secretory phenotype.

To test the effect of Tfam knockdown in VSMCs in vivo, Tfam ^flox/flox mice were crossed with mice expressing the Cre ^ERT2 fusion protein under the smooth muscle myosin ( ^Myh1 ) promoter (Myh11-Cre ERT2). Analysis after 28 weeks of tamoxifen (Tmx) treatment confirmed effective abrogation of Tfam expression in aortas from Myh11-Cre ^ERT2 Tfam ^{fiox/flox mice} , but not in Myh11-Cre ^ERT2 Tfam' ⁷ ' ⁷¹⁷ ' ⁷ mice. ^'71 (SM-Tfam ' ⁷ ' and SM-Tfam ^+/+ mice, respectively), treated with tamoxifen (FIG. 3A). Tfam deletion was accompanied by lower mtDNA content (Figure 3A). Half-life analysis showed a significant decrease in survival with 100% mortality in SM-Tfam'' mice before 33 weeks after tamoxifen treatment (FIG. 3B). Longitudinal analysis of the vascular phenotype revealed a lower blood pressure along with a larger aortic diameter (Fig 3C and D). Histological analysis of SM-Tfam' ⁷ ' mice 28 weeks after tamoxifen treatment revealed aortic dissections, intramural hematomas (IMH), and medial degeneration with elastin lamina degradation and proteoglycan accumulation in the ascending aorta.

To assess how SM-Tfam'' mice respond to a hypertensive test, AngII was infused into SM-Tfam' ⁷ ' and SM-Tfam ^{+ +} mice 56 days after tamoxifen treatment (FIG. 3E). Blood pressure analysis showed a mild increase in SM-Tfamr ⁷ ' mice (Fig. 3F). In addition, ultrasound showed a rapid increase in the diameter of the aortae. 27 ascending (AsAo) and abdominal (AbAo) of SM-Tfanr' mice, supporting the predisposition of these mice to develop aortic aneurysms. More importantly, treatment of S M-Tf a rrr' with AngII produced rapidly fatal aortic aneurysms and aortic dissections, reducing median survival (Fj. 3G). Post-mortem analysis revealed the presence of intramural hematomas (IMH) and aortic ruptures with hemothorax or hemoabdomen (Fig. 3H). Histologic analysis of thoracic and abdominal aortic sections showed increased aortic diameter, aortic dissections, MIH, false lumen formation, and features of medial degeneration, including elastic fiber fragmentation and vascular architecture demonstrating that decline in mitochondrial respiration in VSMCs induce lethal aortic aneurysms and dissections in Marfan animal models.

Example 3: Enhancement of mitochondrial metabolism restores contractility in VSMCs having hereditary aortic aneurysm mutations.

NAD+ is a cofactor for several enzymes with critical roles in mitochondrial activity and metabolic health, and supplementation with NAD+ precursors has been proposed as a strategy to improve mitochondrial functions in several diseases related to mitochondrial decline (Verdin et al. , Science, 2015. 350(6265): pp. 1208-13, Prolla et al., Cell Metab, 2014. 19(2): pp. 178-80). To investigate the therapeutic potential of an enhancement of mitochondrial metabolism with NAD+ in hereditary aortic aneurysms, shFbn1-CM\_\/s were treated with nicotinamide hboside (NR). NR increased OCR and decreased lactate production in shFbn 1-VSMCs to levels found in control shFbnl-VSMCs (Fig. 6A and B). Previous studies have indicated that NR controls mitochondrial metabolism by increasing the expression of Ppargda and Tfam through sirtuin activity. Exposure of shFbn1-CM\_\/s to NR for 5 days increased the expression of Ppargla and Tfam showing a correlation with an increase in mtDNA and the expression of mtDNA encoding the Mt-Co1 transcript (Fig. 6C, D). . Treatment of shFbn 1-VSMCs with NR also increased the fluorescence intensity of polymehzada actin (F-actin), an index of VSMC contractility, while it decreased the expression and activity of the pro-remodeling matrix metalloproteinases Mmp9 and Mmp2. and the pro-fibrotic genes Spp1 and Collagenl (CoHaT) (Fig. 6E), while restoring the mtDNA content and mRNA levels of MT-CO1, MT-ND6, TFAM, and HIF1A (Fig. 6I ). Additionally, NR treatment reduced the expression of collagen extracellular matrix (ECM) (COL1A1) and aggrecan (A CAN) proteins and of the pro-fibrotic factors TGFB3 and connective tissue growth factor (CNN2) ( Fig. 6J). 28

To explore whether mitochondrial metabolism is also affected in other hereditary aortic aneurysms, models of other hereditary aortic aneurysms were obtained by overexpression of disease-causing mutations in primary VSMCs. To mimic familial forms of thoracic aneurysms and dissections and Loeys-Dietz syndrome, lentiviral vectors were used to overexpress Acta2 ^R179H and TGFBR2 ^G357W , respectively. Overexpression of Acta2 ^R179H and TGFBR2 ^G357W reduced OCR, mtDNA content, and mRNA expression of mitochondrial regulators, while increasing extracellular lactate levels (Fig. 7A-C), suggesting that aortic aneurysm mutations force a predominant use of glycolysis as a source of cellular energy. Incubation of cells with NR reversed the effect of both mutations for these metabolic parameters (Fig. 7A-C). In addition, the use of NR also allowed to increase the contractility, measured by the polymerization of F-actin while it produced a decrease in the expression and reduced the activity of matrix metalloproteinases and other secretion markers (Fig. 7D). These data indicate that in VSMCs affected by genetic aortic aneurysms, there is a change in the metabolism of OXPHOS towards glycolysis, and that the improvement of the mitochondrial metabolic state in vitro restores its contractility phenotype.

Example 4: Nicotinamide Riboside Treatment Reverses Aneurysm in a Mouse Model of Marfan Syndrome

The therapeutic potential of NR to prevent and reverse the development of aneurysms in Marfan syndrome model mice by modulating their mitochondrial metabolism was evaluated. 16-week-old Fbn1 ^c1039G/+ mice received intraperitoneal injections of NR every other day for 28 days (Fig. 5A; Fig. 8A). Aortic dilatation and blood pressure completely normalized after 7 days of treatment in male and female mice (Fig. 5B, Fig. 8B). In addition, NR treatment was able to reverse histological characteristics of degeneration in this Marfan syndrome model, such as medial thickness, elastic fiber fragmentation, proteoglycan deposition, and actin polymerization (Fig. 5C and Fig. 8C). NR treatment also restored Tfam mRNA expression and Mt-Co1 mtDNA content to normal levels (Fig. 5D and E).

To characterize the effect of NR treatment at the molecular level, RNA sequencing was performed on aortas from Marfan syndrome model mice and healthy control mice. Hierarchical clustering and transcriptomic analysis revealed that NR treatment reversed the transcriptional changes observed in Fbn1 ^c1039G/+ aortas, changing 29 the expression levels to levels closer to those of the control than to those of Pb _initio39G /+ E mice | NR treatment increased the expression levels of some transcription factors involved in the maintenance of the contractile phenotype and genes related to the smooth muscle contraction apparatus, such as smooth muscle actin (Acta2) and calponinal (Cnn1) ( Fig5F). Thus, enhancement of mitochondrial functions with NR rapidly restores the transcriptional signature and contractile properties of aortic smooth muscle in Marfan syndrome model mice and completely reverses aortic wall remodeling, aortic dilatation, and medial degeneration.

Claims

1 . Nicotinamide adenine dinucleotide (NAD+) precursor, for use in the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology (HAA) characterized by the presence of impaired mitochondrial respiration.

2. Nicotinamide adenine dinucleotide precursor for use according to claim 1, wherein the aortic aneurysm of hereditary etiology (AAH) is selected from the group consisting of: Marfan syndrome, Loeys-Dietz syndrome, Ehlers syndrome -Danlos of vascular type, Cutis laxa syndrome and Familial forms of Non-syndromic Thoracic Aneurysms and Dissections.

3. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 or 2, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is selected from: niacin (NA) or nicotinic acid; nicotinamide (NAM); nicotinamide riboside (NR); nicotinamide mononucleotide (NMN); nicotinic acid mononucleotide; and nicotinic acid adenine dinucleotide; or combinations thereof.

4. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is niacin (NA) or nicotinic acid.

5. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is nicotinamide (NAM).

6. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is nicotinamide riboside (NR).

7. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is nicotinamide mononucleotide (NMN). 31

8. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is nicotinic acid mononucleotide.

9. Nicotinamide adenine dinucleotide precursor for use according to any of claims 1 to 3, wherein the nicotinamide adenine dinucleotide (NAD ⁺ ) precursor is nicotinic acid adenine dinucleotide.

10. Pharmaceutical composition comprising at least one precursor of nicotinamide adenine dinucleotide (NAD+) and at least one pharmaceutically acceptable excipient, for use in the prevention of the appearance and/or treatment of an aortic aneurysm of hereditary etiology (HAA) that occurs characterized by the presence of impaired mitochondrial respiration.

11. Pharmaceutical composition for use according to claim 10, wherein the hereditary aortic aneurysm (HAA) is selected from the group consisting of: Marfan syndrome, Loeys-Dietz syndrome, Ehlers-Danlos syndrome of vascular type, Cutis laxa syndrome and Familial forms of non-syndromic Thoracic Aneurysms and Dissections.

12. Pharmaceutical composition for use according to any of claims 10 or 11, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is selected from the group consisting of: niacin (NA) or nicotinic acid; nicotinamide (NAM); nicotinamide riboside (NR); nicotinamide mononucleotide (NMN); nicotinic acid mononucleotide; and nicotinic acid adenine dinucleotide; or combinations thereof.

13. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is niacin (NA) or nicotinic acid.

14. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide (NAM). 32

15. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide riboside (NR).

16. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide mononucleotide (NMN).

17. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid mononucleotide.

18. Pharmaceutical composition for use according to any of claims 10 to 12, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid adenine dinucleotide.

19. Nutraceutical composition comprising at least one nicotinamide adenine dinucleotide (NAD+) precursor and at least one nutraceutically acceptable excipient, for use in preventing the appearance of an aortic aneurysm of hereditary etiology (HAA) characterized by the presence of poor mitochondrial respiration.

20. Nutraceutical composition for use according to claim 19, wherein the hereditary aortic aneurysm (HAA) is selected from the group consisting of: Marfan syndrome, Loeys-Dietz syndrome, Ehlers-Danlos syndrome of vascular type, Cutis laxa syndrome and Familial forms of non-syndromic Thoracic Aneurysms and Dissections.

21. Nutraceutical composition for use according to any of claims 19 or 20, wherein the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is selected from the group consisting of: niacin (NA) or nicotinic acid; nicotinamide (NAM); nicotinamide riboside (NR); nicotinamide mononucleotide (NMN); nicotinic acid mononucleotide; and nicotinic acid adenine dinucleotide; or combinations thereof.

22. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is niacin (NA) or nicotinic acid. 33

23. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide (NAM).

24. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide riboside (NR).

25. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinamide mononucleotide (NMN).

26. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid mononucleotide.

27. Nutraceutical composition for use according to any of claims 19 to 21, in which the precursor of nicotinamide adenine dinucleotide (NAD ⁺ ) is nicotinic acid adenine dinucleotide.