WO2024218149A1 - Treatment, prognosis and/or diagnosis of aortic stenosis - Google Patents

Abstract

The present invention refers to a method for the treatment, prognosis and/or diagnosis of aortic stenosis (AS), preferably in patients with diabetes mellitus (DM).

Description

TREATMENT, PROGNOSIS AND/OR DIAGNOSIS OF AORTIC STENOSIS

FIELD OF THE INVENTION

The present invention belongs to the medical field. Specifically, the present invention refers to a method for the treatment, prognosis and/or diagnosis of aortic stenosis (AS), preferably in patients with diabetes mellitus (DM).

STATE OF THE ART

DM and AS are chronic diseases that frequently occur in the same patient and that have an important relationship. Numerous studies have shown that the prevalence of DM is higher in patients with AS and that DM increases the severity of AS. When both pathologies concur in the same patient, the treatment of AS becomes more complex, and if they remain untreated these pathologies provoke significant morbidity and mortality. The recent worldwide increase in the incidence of obesity is associated with an increase in DM, and the progressive aging of the population has been associated with an increase in DM and AS. Consequently, there is likely to be an increase in the frequency with which these two pathologies coincide in patients in the future.

AS is the valve disease that most frequently requires a surgical or other type of intervention in developed countries. AS prevents the aortic valve (AV) from opening properly and the ensuing narrowing of the aortic valve restricts the flow of blood, preventing part of the blood from the left ventricle from passing to the rest of the body. The most common form of AS is degenerative AS (DAS) in which age-related calcium deposition stiffens the valve and restricts its opening.

DAS develops progressively and its early inflammatory phases are very similar to atherosclerosis with which it shares risk factors. There is clinical and histological data indicating that far from reflecting passive degeneration, DAS is an active process in which lipoprotein deposition, chronic inflammation and the osteoblastic transformation of interstitial cells in later phases are associated with the accumulation of calcium in the valve tissue, which produces a calcified AS. The progression of DAS is fundamentally regulated by valve interstitial cells, the most abundant cell type in the AV. Valve interstitial cells adopt different phenotypes in calcified AVs that vary in their degree of activation, and some of these are similar to the myofibroblasts and osteoblasts responsible for calcium deposition. The accumulation of calcium increases in stiffness and narrowing of the AV, affecting haemodynamics. The pressure exerted on the AV wall increases dramatically as DAS progresses and this is considered a diagnostic parameter to define the stage of the disease, along with valve narrowing.

High blood pressure, hypercholesterolemia and DM are among the factors known to exacerbate the progression of atherosclerosis and DAS. Both type 1 DM (DM1) and type 2 DM (DM2) accelerate atherosclerosis, not only due to the increase in blood glucose levels but also, due to the insulin resistance and dyslipidemia associated with these diseases. Specifically, DM2 is associated with a marked inflammatory response and enhanced lipid accumulation, which is generally associated with a deterioration in cardiac function. These mechanisms also affect the development of DAS, since they are associated with hypertrophic remodelling of the left ventricle, a greater left ventricular mass and dampened systolic function. These processes are the main mechanisms through which DM2 increases the cardiac risks associated with AS. Evidence that DM2 is a risk factor for DAS came from a study carried out in a Swedish population in which the incidence of DAS was 34% more frequent in patients with DM2 (3.42 %) than in non-diabetics (1.68%, p<0.05). In Spain, the incidence of AV replacement was 2.6 times higher in patients with DM2 than in non-diabetics (IRR 2.60; 95% CI 2.56-2.65).

DAS usually remains asymptomatic for long periods (up to several years) but once diagnosed, its progression is inevitable, rapid and with a poor prognosis. Therefore, finding new therapeutic options to treat this disease represents an important challenge. Since DM influences the survival of patients with DAS, patients should be stratified based on whether or not they have DM in order to adapt the treatment strategy accordingly. Furthermore, since no parameters adequately predict the evolution of the disease, all available information must be considered in clinical decision-making. It is important to note that DM2 is a progressive disease which, despite causing significant damage to numerous tissues and contributing to the development of multiple diseases (including AS), can remain unnoticed during its initial stages.

Despite much effort to find effective treatments for DAS, the only effective treatment currently available is AV replacement through a surgical or transcatheter intervention. Recent evidence suggests that surgical intervention is appropriate for patients with severe asymptomatic AS, although DM2 is an important risk factor in patients with DAS who are to undergo surgery as it affects both the survival and quality of life (QoL) of these patients. Therefore, DM2 patients should undergo regular medical, electrocardiographic and echocardiographic check-ups in order to monitor the evolution of DAS, and to define the most appropriate time and strategy for treatment. In terms of pharmacological treatments, despite studies with statins, and drugs related to calcium and phosphate metabolism, no therapies are currently capable of slowing down or altering calcification. However, valve calcification has been related to immunological phenomena that favour mineralization of the AV’s interstitial cells, such that treatments targeting the immune response in DAS could be of interest to combat this chronic process. Regarding specific therapies aimed at diabetic patients with DAS, oral antidiabetic drugs or insulin targeting the AV or myocardium could be of interest. In theory, such treatments could slow the progression of DAS, reducing the hemodynamic impact on left ventricular function and remodelling, and consequently improve clinical outcomes.

Therefore, there is an unmet medical need to achieve a treatment and diagnosis of AS, particularly in patients with DM, such that patients who have these pathologies can receive personalized treatment. The present invention aims to resolve this issue and provide a new treatment and in vitro method for the prognosis and/or diagnosis of AS in patients, particularly in patients with DM.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention refers to a method that allows AS to be diagnosed, particularly in patients with DM, so that patients who have both pathologies can receive personalized treatment. Specifically, the present invention is an in vitro method for the prognosis and/or diagnosis of AS, preferably in patients with DM.

In particular, the inventors have identified a possible marker for the diagnosis of DM in patients with DAS, the APOC2 protein. The expression of APOC2, or the APOC2 gene that encodes this protein, is altered in biological samples from patients with DAS and DM (DAS + DM) relative to patients with DAS alone.

Specifically:

The levels of APOC2, as well as those of the transcripts that encode said protein, are reduced in AV tissue from patients with DAS + DM relative to tissue from patients with DAS alone (Figure 1).

The APOC2 in the plasma from patients with DAS + DM is greater than that in plasma from patients with DAS alone (Figure 2). There is less APOC2 in AV interstitial cells treated with plasma from patients with DAS + DM relative to those treated with plasma from patients with DAS alone (Figure 4).

Results obtained with the in vitro model show that there is a tendency towards a decrease in the expression of APOC2 by AV interstitial cells when treated with plasma from diabetic patients (Figure 4) in the days prior to calcification in the model (calcification occurs from day 7 onwards, Figure 3). For this reason, the APOC2 protein is postulated as a protein with predictive value for the severity of AS, particularly in patients with DM. Indeed, this protein could be involved in the onset of calcification since differences in its expression were found in the days prior to calcification in the cell model and these differences are apparent in severe states of the pathology (validation in plasma samples).

These results suggest a potential use of APOC2 as a marker that discriminates between patients with DAS + DM and patients who have DAS alone. Note that the expression of this protein varies depending on the biological samples analysed. This is probably because the APOC2 protein is secreted into the plasma, such that high levels in the plasma are usually associated with poor tissue retention of APOC2, and vice versa.

On the other hand, Example 2.4 and Figure 6 shows that APOC2 can be used as a therapeutic target for the treatment of AS. According to these results, when APOC2 was silenced, calcification is less intense. In fact, when VICs were cultured in MO after the treatment with siRNA, levels of calcification were the same than in the FIB media. These results were confirmed also in the culture media where calcium levels were also measured. So, although a siRNA has been used as proof of concept in the present invention, any APOC2 inhibitor could be used in the context of the present invention, since the contribution that the present invention makes over the prior art is that APOC2 can be used as a therapeutic target for the treatment of AS, preferably in patients suffering from DM.

Thus, the present invention initially refers to an in vitro method for the diagnosis and/or prognosis of AS, preferably in patients with DM. The invention involves a method to determine the level of APOC2 protein or APOC2 gene expression in a biological sample obtained from a patient, whereby a deviation in expression with respect to a previously established reference value is indicative that the patient with DM suffers AS.

A second aspect of the present invention refers to an in vitro method to predict whether a patient with DM can develop AS. This aspect of the invention involves a method to determine the level of APOC2 protein or APOC2 gene expression in a biological sample obtained from a patient, whereby the deviation in expression with respect to a previously established reference value is indicative that the patient with DM can develop AS.

A third aspect of the present invention refers to an in vitro method to determine if a patient with DM could benefit from a treatment for AS. The invention involves a method to determine the level of APOC2 protein or APOC2 gene expression in a biological sample obtained from a patient, whereby a deviation in expression with respect to a previously established reference value is indicative that a patient with DM could benefit from treatment for AS.

In a preferred aspect of the present invention the biological sample is plasma, blood, serum or tissue from a heart biopsy.

In a preferred aspect, the present invention comprises a method to determine the value of the APOC2 protein or APOC2 gene in a biological sample of plasma, blood or serum obtained from a patient, where an expression greater than a previously established reference value is indicative that the patient with DM has AS, that the patient with DM can develop AS, or that the patient with DM could benefit from treatment against AS.

In a preferred aspect, the present invention comprises a method to determine the value of the APOC2 protein or APOC2 gene in a biological tissue sample from a heart biopsy, whereby expression lower than a previously established reference value is indicative that the patient with DM has AS, that the patient with DM can develop AS, or that the patient with DM could benefit from treatment against AS.

The fourth aspect of the present invention refers to the in vitro use of the APOC2 protein or APOC2 gene, alone or in combination, for the diagnosis and/or prognosis of AS in patients with DM, to predict whether a patient with DM can develop AS, or to determine if a patient with DM could benefit from treatment against AS.

The fifth aspect of the present invention refers to the in vitro use of a kit consisting of reagents to measure the expression of the APOC2 protein or APOC2 gene, for the diagnosis and/or prognosis of AS in patients with DM, to predict whether a patient with DM can develop AS, or to determine if a patient with DM could benefit from treatment against AS.

The sixth aspect of the present invention refers to antidiabetic drugs, or any composition comprising such a drug, for use in a method for the treatment of patients with AS, wherein the method comprises to determine whether a patient may benefit from treatment with antidiabetic drugs.

The present invention also refers to a method to detect APOC2, or the transcripts that encode said protein, in a sample from a patient with DM who could also have AS, wherein the method comprises bringing the biological sample into contact with a reagent capable of specifically detecting APOC2 or the transcripts that encode said protein and enabling the levels of said biomarker to be measured in the sample.

In a preferred form of the invention, AS is DAS.

In a preferred form of the invention, DM is type 2 DM.

In a preferred form of the invention, AS is DAS stenosis and DM is type 2 DM.

In a preferred aspect of the present invention, the antidiabetic drug is a drug selected from the list made up of metformin, sodium-glucose cotransporter 2 inhibitors, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors or glucagon-like peptide-1 receptor agonists.

In a preferred form of the invention, AS can be confirmed through an imaging technique such as: echocardiogram, Doppler ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). For example, in prospective studies performed in the laboratory in the context of the present invention, Doppler ultrasound (3D-mode and Doppler echocardiography) or MRI were performed.

In a preferred form of the invention, DM can be confirmed by a device for measuring glucose or insulin levels in plasma, serum, blood or in any biological sample, such as a glucometer or a biosensor of glucose/insulin. The glycated/glycosylated haemoglobin test could also be used.

The present invention also relates to a computer-implemented invention in which a processing unit (hardware) and software are configured to: a) receive values regarding APOC2 concentrations or APOC2 expression; b) process the received values of concentration or expression to find substantial variations or deviations; and c) provide an output through a terminal display of the variation or deviation in the concentration, whereby the variation or deviation of the concentration may indicate that the subject could be developing AS depending on the parameters used.

The present invention also relates to a computer program or a computer-readable medium containing means to apply a method as defined in any of the above claims. Moreover, the present invention also refers to:

APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use in a method for the treatment of AS. Alternatively, the present invention also refers to a method for treating AS which comprises the administration of a therapeutically effective amount of an APOC2 inhibitor, or pharmaceutical composition comprising thereof.

In a preferred embodiment, the present invention refers to the treatment of AS in patients suffering from diabetes, preferably DM.

In a preferred embodiment, the inhibitor inhibits the expression the APOC2 gene, or it carries out a pharmacological inhibition.

In a preferred embodiment, the inhibitor is an RNA molecule preferably selected from the group consisting of a small interfering RNA molecule (siRNA), an anti-sense RNA molecule, a microRNA molecule (miRNA) and/or a short hairpin RNA (shRNA), and/or a genome editing tool preferably selected from the group consisting of zinc-finger proteins (ZFNs), transcription activator-like effector nucleases (TALENs) and/or CRISPR/Cas.

In a preferred embodiment, the inhibitor is a siRNA characterized by a nucleotide sequence complementary to the gene APOC2 and that interferes with the expression of the gene APOC2 (Ensembl:ENSG00000234906 MIM: 608083; AllianceGenome:HGNC:609; Gen ID: 344).

The present invention also refers to an in vitro method for monitoring the efficacy of a treatment and/or for predicting the response to a treatment in a patient suffering from AS which comprises: (a) Measuring the level of expression or activity of APOC2 in a biological sample isolated from the patient after the treatment; (b) wherein if the level of expression or activity of APOC2 determined in step (a) is statistically lower than the level of APOC2 before the treatment, it is indicative that the patient is responding to the treatment.

The present invention also refers to an in vitro method for identifying and/or producing candidate compounds useful in the treatment of AS which comprises: (a) Measuring the level of expression or activity of APOC2 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if the level of expression or activity of APOC2 determined in step (a) is statistically lower than the level of APOC2 determined before the administration of the candidate compound, is indicative that the candidate compound is effective in the treatment of AS. The present invention also refers to an in vitro method for the diagnosis and/or prognosis of AS, preferably in patients with diabetes, most preferably DM, which comprises, a) determining the level of expression of APOC2 protein, or APOC2 gene, in a biological sample obtained from a patient, b) wherein a deviation in expression from a pre-established reference value is indicative that the patient has AS or that the patient with diabetes has AS.

In a preferred embodiment, the biological sample is plasma, blood, serum or tissue from a heart biopsy.

In a preferred embodiment, the present invention comprises a) determining the level of expression of APOC2 protein, or APOC2 gene, in a biological sample of plasma, blood or serum obtained from a patient, b) wherein a level of expression above a previously established reference value is indicative that the patient has AS, or that the patient with diabetes may develop AS.

In a preferred embodiment, the AS is DAS.

In a preferred embodiment, the DM is type 2 DM.

The present invention also refers to the in vitro use of APOC2 protein, or the APOC2 gene, or of a kit comprising reagents for the measurement of APOC2 protein expression levels, or the APOC2 gene, for the diagnosis and/or prognosis of AS, preferably in patients with diabetes, or to predict whether a patient with diabetes may develop AS.

In the context of the present invention, the following terms or expressions are defined for a better interpretation of its scope:

• The term "comprising" means including but not limited to that which follows the word "comprising." Thus, the use of the term "comprising" indicates that the elements listed are necessary or mandatory but that other elements are optional and may or may not be present.

• The term "consisting of means that it includes, and is limited to, what follows the phrase "consisting of. Therefore, the phrase "consisting of indicates that the elements listed are necessary or mandatory, and that no other elements may be present.

• The term “antidiabetic drug” refers to any drug aimed at reducing the abnormally high blood glucose levels in patients with DM, increasing insulin sensitivity and/or stimulating the release of insulin from the pancreas. The major classes of antidiabetic drugs are biguanides, sulfonylureas, meglitinides, thiazolidinediones (TZDs), dipeptidyl peptidase-4 (DPP -4) inhibitors, sodium-glucose cotransporter 2 (SGLT2) inhibitors and alpha-glucosidase, preferably: Metformin; SGLT2 inhibitors; TZDs; sulfonylureas; DPP -4 inhibitors or glucagon-like peptide-1 receptor agonists.

• The term “AS” refers to a disease that affects the AV, restricting the flow of blood. It occurs when the AV does not open properly, preventing part of the blood from the left ventricle from passing to the rest of the body. The most common form of AS is DAS, which occurs through to aging due to the accumulation of calcium deposits that harden the valve and restrict its opening. Treatment of AS usually involves valve replacement through cardiac surgery.

• According to the present invention, a "pre-established reference value" or a "cut-off value can be determined experimentally, empirically or theoretically. According to the present invention, the "pre-established reference value" refers to a value previously determined in control subjects. A "pre-established reference value" may also be arbitrarily selected based on existing experimental and/or clinical conditions, as could be recognised by a standard specialist in the field. The "threshold value" must be determined to obtain optimal sensitivity and specificity according to the performance of the test and the benefit/risk equilibrium (clinical consequences of false positives and false negatives). Normally, the optimal sensitivity and specificity (and therefore the "threshold value") can be determined through a Receiver Operating Characteristic (ROC) curve based on experimental data.

• The expression “therapeutically effective amount” of a composition comprising APOC2 inhibitors is intended an amount that, when administered as described herein, brings about a positive therapeutic response in a subject having aortic stenosis. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

Description of the figures

Figure 1. A) The expression of the APOC2 protein or APOC2 gene obtained by transcriptomic and proteomic analysis of AV tissue from DAS patients with DM or without DM (nDM). B) A correlation analysis between the quantitative gene expression and the protein product: S= Spearman correlation coefficient. Figure 2. Validation of the APOC2 in plasma samples from patients with DAS, with (DM) or without DM (nDM). A) Validation using ELISA. B) Validation by turbidimetry.

Figure 3. Confirmation of calcification in the in vitro model. A) Expansion of valve interstitial cells (VICs). B) Visualization and C) quantification of the calcium deposition through alizarin red staining, and D) the quantification of BMP -2 in western blots of VICs treated with FIB or osteogenic medium for 7 days: FIB, special medium for fibroblasts; MO, osteogenic medium.

Figure 4. A) Analysis of APOC2 expression by valve interstitial cells (VICs) treated with plasma from patients with DAS, with (DM) or without DM (nDM). B) Confirmation of the accumulation of APOC2 in western blots.

Figure 5. Representative scheme. The data obtained through the in vitro model show that there is a tendency for the valve interstitial cells to decrease in number following treatment with plasma from diabetic patients (Figure 4) in the days prior to calcification in the model (calcification occurs from day 7: Figure 3). Therefore, it is postulated that the APOC2 protein has predictive value for the severity of AS in patients with DM and that it could be involved in the onset of calcification since differences in expression were found in the days prior to calcification in the cell model. In addition, differences in the amounts of this protein appear in severe states of the pathology (validation in plasma samples).

Figure 6. Effect in the in vitro model of the treatment with recombinant APOC2 (A, B, C) and, the silencing with siRNA APOC2 (D, E, F). A) Quantification of the calcium deposition through alizarin red staining, B) quantification of the secreted calcium in the culture media and C) correlation between calcium deposition and secretion after treatment with APOC2. D) Quantification of the calcium deposition through alizarin red staining, E) quantification of the secreted calcium in the culture media and F) correlation between calcium deposition and secretion after silencing with siRNA APOC2.

Detailed description of the invention

The present invention is illustrated by the Examples set out below without any intention to limit the scope of protection. Example 1. Materials and Methods

Example 1.1 Selection of subjects for the study

Aortic valve tissue and plasma samples were obtained from patients diagnosed with DAS with or without DM2 who underwent AV replacement surgery. The inclusion criteria established were: over 50 years of age, not having participated in a clinical trial with drugs at the time of inclusion, diagnosis of valve involvement or absence thereof, with or without DM, and having signed their informed consent (if an individual was unable to provide consent, their legal representative was authorised to provide consent on their behalf).

The exclusion criteria for all patients included: refusal to provide signed informed consent, a severe co-morbidity (ischemic heart disease with ventricular dysfunction, end-stage chronic kidney disease -stage 5 or dialysis, familial hypercholesterolemia, secondary AHT, cancer, a bicuspid AV, a family or personal history of an aortopathy, moderate or more severe rheumatic or mitral valve disease.

The study was carried out in accordance with the Helsinki Declaration and having received a favourable evaluation from the Ethical Committees of the hospitals involved in obtaining samples.

Example 1.2. Sample collection

To obtain plasma, blood samples were collected from all the participants in tubes pretreated with ethylenediaminetetraacetic acid (EDTA) and they were processed in less than 2 hours from collection to avoid deterioration. The blood was centrifuged at 1125g (5804R, Eppendorf) for 10 min at 4 °C and the plasma was stored in 500 pl aliquots at -80 °C until use.

Aortic valves were processed within a maximum of 2 hours from extraction. They were first washed with a saline solution to eliminate any possible traces of blood and the 3 leaflets were then stored at -80 °C until use.

Example 1.3. Preparation of tissue samples for proteomic analysis

Calcified leaflets and leaflets without calcification were selected from the same patient and the samples were lysed with stainless steel balls in a grinder, quantifying the amount of protein recovered using the Pierce micro BCA protein quantification assay (Thermo Scientific). To this end, the samples were introduced into 1.5 ml tubes containing 3.2 mm stainless steel balls (Eppendorf Navy kits) and 400 pl of a lysis solution made up of 4.44% SDS and 0.1 M Tris diluted 50% in water. The tubes were placed in the grinder and crushed approximately 3 times until homogenization was complete, after which they were centrifuged for 2 min at 11,000 ref. Subsequently, the supernatants were collected in 1.5 ml tubes and they were subjected to 5 sonication-ice cycles of 1 min. The lysates were then incubated for 10 min at 95 °C in a thermoblock with agitation at 600 rpm, and then left for 5 min at room temperature (RT). The sonication-ice cycles were then performed again, and the samples centrifuged for 15 minutes at 16,000 xg at 15 °C to eliminate the insoluble material. Finally, the supernatants were transferred to a new 1.5ml tube, and the samples were aliquoted and stored at -40 °C until quantification and their further analysis. The protein was quantified in duplicate at dilutions of 1/100 and 1/1000, titrating against a standard curve of BSA prepared in the lysis solution used for tissue protein extraction.

Example 1.4. Tandem Mass Tag™ (TMT) Isobaric Marking System

Protein from each sample (80 pg) was digested using the FASP (Filter-Aided Sample Preparation) protocol. First, the samples were boiled in the presence of dithiothreitol (DTT, 100 mM) and solubilized in a urea buffer (8M Urea, 100 mM Tris-HCl pH=8.5). After loading the samples onto the filter and washing several times with urea buffer, the thiol residues of the proteins were carboxymethylated for 20 min at RT in the dark using iodoacetamide. Excess reagent was removed with successive washes with urea buffer and triethylammonium bicarbonate (TEAB, 20 mM), and then recovered by centrifugation at 14,000 ref for 10 min at RT. The protein extracts retained on the filter were incubated with shaking for 18h at 37 °C with a trypsin solution (20 mM) prepared in TEAB at a ratio of 3: 100 (trypsimprotein). The following day, the resulting peptides were recovered by adding a TEAB solution (20 mM) and centrifuging at 14,000 ref for 15 min at RT. The peptides were then dried with a Savant SPD11 IV vacuum centrifuge (Thermo Scientific) and the efficiency of digestion was assessed for each sample by liquid chromatography linked to tandem mass spectrometry (LC-MS/MS). The samples were labelled using two commercial Tandem Mass Tag™ (TMT) 10-plex kits (Fisher Scientific) according to the manufacturer's instructions.

Example 1.5. Tissue analysis by mass spectrometry (MS)

For labelling, each of the samples were reconstituted in 100 pl of TEAB (20 mM) and incubated for 10 min at 20 °C with shaking. Subsequently, they were labelled with the corresponding TMT label for 1 h at RT, stopping the reaction by adding 8 pl of 5% hydroxylamine and incubating for 15 min. The labelled peptides from each patient were mixed and dried under vacuum, and finally 800 pg of these labelled peptides were diluted in 400 pl of 5 mM ammonium formate [pH 10] and 2% acetonitrile (ACN). Half of the peptide mix was fractionated by high pH reversed-phase chromatography on XBridgePeptide BEH C18 columns (Waters) using a flow rate of 200 pl/min: Solvent A - 5 mM ammonium formate [pH 10] and 2% ACN; Solvent B - 5 mM ammonium formate [pH 10] and 90% ACN. Fractions were collected every 2 min from minute 10 to minute 80, considering 7 fractions recovered between minute 16 and 60 for subsequent analysis.

The labelled peptides recovered were analysed on an Orbitrap Fusion Lumos™ Tribrid mass spectrometer (Thermo Scientific) equipped with a Dionex Ultimate 3000 ultra-high pressure coma system (Thermo Scientific) and using an Advion Tri Versa NanoMate nanospray interface (Advion Inc. Biosciences). Peptides were loaded onto a p-pre-column (300 pm id x 5 mm, C- 18 PepMaplOO, 5 pm, 100 A, C18 Trapcolumn: Thermo Scientific) with a flow rate of 15 pl/min and separated on a C-18 analytical column (NanoEase MZ HSS T3 column - 75 pm x 250 mm, 1.8 pm, lOOA: Waters) at a flow of 250 nl/min. The analytical separation lasted 210 min and followed three consecutive linear gradient steps: 3-35% solvent B over 180 min, 35- 50% solvent B over 5 min, and 50-85% solvent B over 2 min. This was then followed by isocratic elution at 85% solvent B for 5 min and stabilization to the initial conditions: solvent A = 0.1% formic acid in water, solvent B = 0.1% formic acid in CH3CN.

The mass spectrometer analysis was performed in data-dependent acquisition mode. In each data collection cycle, a full scan (400-1600 m/z) was acquired in Orbitrap (1.2 x 105 resolution and 2x 105 Automatic gain control -AGC). The most abundant ions were selected by fragmentation using Higher-energy Collisional Dissociation (HCD) with a collision energy of 30%, 0.25 Q activation and an AGC of 1 xlO⁴, an isolation window of 1.6 Da, a maximum ion accumulation time of 50 ms and a fast ion scanning rate. Dynamic exclusion parameters were set to 1 repetition for 30 s. The spray voltage on the NanoMate source was programmed at 1.60 kV and the radio frequency was tuned to 30%. The minimum signal required to trigger the switch from MS to MS/MS was set at 5,000. The mass spectrometer worked in positive polarity mode and single-charge precursors were rejected by fragmentation. Example 1.6. Proteomic data processing

The spectra to identify proteins were analysed with the Proteome Discoverer version 2.3 software package using SEQUEST-HT (Thermo Fisher) to screen against the SwissProt Human database (April 2020). The selected search parameters were: trypsin digestion with at most two missed cleavage sites, carbamidomethylcysteine and TMT-lOplex modifications at N-terminal and Lys residues as fixed modifications, and the oxidation of methionines and acetyl at the N-terminal residues as a variable modification. The mass tolerance of the precursors and fragments was 20 ppm and 0.02 Da, respectively. Peptide identification was validated using a false discovery rate (FDR) of 1% calculated against the inverted databases.

The quantitative analysis involved 4 conditions (n=4 per group): DM-Cal, DM-nCal, nDM- Cal, nDM-nCal. Samples were paired by patient, with or without calcification (Cal and nCal), and the intensities of the reporter ions were used for protein quantification. Unique peptides (peptides not shared with other protein groups) were considered for quantification and statistical analysis. For each TMT experiment, peptide quantification was normalized by summing the abundance for each channel of all peptides identified in the experiment. The channel with the highest total abundance was taken as a reference and the abundances were corrected in all the other channels applying a constant factor, thereby obtaining an equivalent total abundance for all channels. Proteins were quantified by summing the normalized intensities of all the peptides of a protein.

The data were transformed to a logarithmic scale to apply a linear model and the TMT experiments were normalized using quantile normalization. The data were filtered to only retain proteins with valid quantifications, at least 3 valid values in a minimum of one group. Missing values were imputed with normally distributed random numbers (centred at -1.8 standard deviation units and within 0.3 units of non-missing values).

Example 1.7. Preparation of tissue samples for transcriptomic analysis

RNA was extracted from the valve tissue samples using the “RNeasy lipid tissue kit” (Qiagen), including a DNase treatment step to eliminate possible DNA contaminants. The yield and purity of the RNA extracted was quantified using Nanodrop equipment. After quality control of the samples, libraries were generated with the TruSeq RNA Access Library Prep kit (TruSeq RNA Access Library Prep Guide Protocol: 15049525 B), and they were prepared for sequencing by random DNA or cDNA fragmentation, followed by incorporation of adapters at the 5' and 3' ends. Alternatively, tagmentation combines the fragmentation and ligation reaction in a single step, which significantly increases the efficiency of library preparation. Subsequently, the adapter-ligand fragments are amplified by PCR and gel purified.

Example 1.8. Transcriptome sequencing using RNA-Seq

Sequencing was carried out using the Illumina Novaseq6000 platform, obtaining paired reads of 150 bases. To generate a set of identical fragments, the library was loaded into a Flow cell, where the fragments are captured on a lawn of oligos linked to the surface that are complementary to the library adapters. Quality control of the sequences was achieved using the FastQC tool, evaluating the distribution of the quality throughout the reads, the GC content, the presence of adapters, and the indices and/or overrepresented sequences. From the information obtained the bases or complete reads that did not meet predetermined requirements were eliminated (trimming). Alignments were made taking the Homo sapiens transcriptome (GRCh38) as a reference. The sequences were pseudo-aligned against the reference transcriptome to directly quantify the transcripts using the Salmon algorithm, taking into account different experimental characteristics and common biases observed in RNA-Seq data. Quantification was represented as TPM (transcripts per million).

Example 1.9. Enzyme-Linked Immunosorbent Assay (ELISA)

Commercial ELISA kits based on the principle of sandwich ELISA in 96-well microplates were used. The ELISA plates were previously coated with the specific antibody for each protein. Initially, 100 pl/well of sample or standard were incubated for 90 min. at 37°C and then the biotinylated secondary antibody specific for each protein was added, in amounts of 100 pl/well for 60 min. at 37°C. After washing the wells 3 times with the washing solution, 100 pl/well of streptavidin-peroxidase enzyme was added and incubated for 30 min. at 37°C. After performing 5 washes, 90 pl of substrate-chromogen was added to each well and incubated for 15 min. at 37°C or until the colorimetric reaction catalysed by the substrate occurred. The reaction was stopped with an acidic solution and the optical density was measured in an Infinite 200 Pro plate reader (Tecan) at a wavelength of 450 nm. The calculation of the concentration of each protein in the samples was carried out by comparing the optical density values of the samples with the standard curve. Both the samples and the different values of the standard curve were performed in duplicate. Example 1.10. Turbidimetry

The turbidimetry tests were carried out in collaboration with the Clinical Analysis Laboratory at the Hospital “12 de Octubre” in Madrid. Briefly, the appropriate amount of each sample was mixed with a diluent solution and the basal absorbance of the sample was measured at a wavelength of 340 nm on an automated A25 system (Biosystems). Subsequently, the corresponding antibody was added to the sample and the turbidity was quantified after a 2 min incubation.

Example 1.11. In vitro model

VICs (Innoprot, Reference P10462) isolated from heart valves and cryopreserved from primary cultures were used in the in vitro model, guaranteeing expansion over 15 population doublings. The cells were cultured at 37 °C and in a humidified atmosphere of 5% CO2 in polylysine (2 pg/cm²: Sigma) coated flasks. When the cells achieved a uniform layer that covered approximately 70% of the surface, they were passaged to 75 cm² culture flasks with a surface filter at a density of 10 xlO⁶ cells/flask. The cells were cultured in FM-2 fibroblast medium (Innoprot) that is designed for the optimal growth of human cardiac fibroblasts in vitro, supplemented with 5% foetal bovine serum (FBS), 1% fibroblast growth factor 2 (FGF2) and 1% penicillin/streptomycin.

The culture medium was replaced 3 times a week and when the cells reached 80-90% confluency, the cells were dissociated with 3 ml of a trypsin-EDTA solution for 2 min at 37 °C. This reaction was stopped by adding 9 ml of neutralization solution (Dulbecco’s modified MEM medium -DMEM- supplemented with 10% FBS), the cells were recovered and transferred to a 15 ml tube to be centrifuged at 290g (5804R, Eppendorf) for 5 min at RT. The cell pellet was resuspended in FM-2 medium and the cells were returned to culture as indicated above until the fourth passage was reached to carry out the experiments.

Once the cells reached the fourth passage, the cells were again dissociated again with trypsin- EDTA but they were then cultured in 10 cm² 6-well plates (Nunc) at a density of 5,000 cells/cm² for 7 days in one of two different culture media to determine the onset of calcification and of fibroblast activation. The media used were a special FIB medium that favours a quiescent fibroblast phenotype and that is composed of DMEM supplemented with 2% FBS, 1% penicillin/streptomycin, 1% glutamine, FGF-2 and insulin 145, or an osteogenic medium (OM) to induce osteogenic differentiation of the VICs that is composed of FIB medium supplemented with 5% ascorbate-2-phosphate and dexamethasone (100 nM). Osteogenic differentiation was subsequently verified by alizarin red staining and by analysing markers of calcification.

Example 1.12. Alizarin Red Staining

The culture medium was removed from the wells, the cells were washed twice with PBS (phosphate buffered saline) and they were then fixed for 15 min with 4% paraformaldehyde. Subsequently, the cells were incubated for 10 min with alizarin red (Sigma Aldrich) and they were then washed with distilled water to remove any excess dye. Calcium deposits were visualized under an Olympus 1X83 inverted microscope, and a 49 images per well were captured and analysed using Scan^AR software. Each experiment was performed in triplicate.

Example 1.13. Treating cells with patient plasma

Once the cells reached the fourth passage, they were dissociated with trypsin-EDTA and cultured in 10 cm² 6-well plates (Nunc) for 5 days to analyse the expression of the proteins identified in the proteomic studies. The cells were cultured in FIB medium supplemented with 5% plasma from DAS patients with or without DM. After the five days exposure to the plasma, protein extracts were analysed and immunodetection assays were performed for the proteins of interest.

Example 1.14. Protein extracts from cell cultures

Protein extracts were obtained from the cells during the 7 days by dissociating the cells with trypsin-EDTA (300 pl/well) for 2 min at 37 °C and stopping the reaction by adding 600 pl/well of neutralization solution. The cells were transferred to a 1.5 ml tube and centrifuged at 290g for 5 min at RT. The supernatant was then discarded, the cells washed twice with 1 ml of PBS and recovered by centrifugation at 290 g for 5 min. The cell pellet was resuspended in 15 pl of lysis solution 1, composed of 2.5 mM DTT, 0.1% SDS and 1 mM PMSF, and then subjected to 3 sonication/ice cycles of 1 min. The lysate was then centrifuged at 19,000g for 10 min at 4 °C and the supernatant (El) collected. Lysis solution 2 (15 pl) composed of 7M urea, 2M thiourea and 4% of 3-[(3-Colamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS) was added to the remaining pellet and the lysate was again subjected to 3 cycles of sonication/ice for 1 min. Finally, this lysate was centrifuged at 19,000g for 10 min at 4 °C and the supernatant obtained (E2) was combined with supernatant El. Once the cell lysates were obtained, the protein concentration was determined using the Bradford-Lowry method (BioRad) and they were stored in 1.5 ml tubes at -80 °C until use. Example 1.15. Immunodetection

One-dimensional electrophoresis of the proteins obtained from the cell extracts was performed on a 15% SDS-PAGE gels in a Miniprotean II vertical electrophoresis system (Bio-Rad), applying an initial voltage of 80 V for 5-10 mins followed by of a constant voltage of 25 mA for 1 hour. The proteins were then transferred semi-dry to a nitrocellulose membrane (TransBlot SD Cell BioRad) applying a voltage of 12 V for 1 h and using a transfer buffer composed of 25 mM Tris, 151.8 mM Glycine and 20% methanol (v/v). The membrane was then stained with a 0.1% solution of Ponceau Red S (Sigma) in 1% acetic acid for 5 min to confirm adequate protein transfer and that all the samples had been loaded equally. The membranes were blocked for 1 h at RT in a solution of PBS/0.1% (v/v) Tween20 containing 7.5% (w/v) skimmed milk powder and they were probed overnight with the corresponding primary antibodies against BMP -2, APOC2 or GAPDH diluted 1 :100, 1 : 100 and 1 : 1000, respectively in wash buffer containing 5% (w/v) skimmed milk powder. The following day, the membranes were washed with shaking 3 times for 10 min with PBS-Tween 0.1% and they were then probed for 1 h. at RT with the corresponding secondary antibody, a peroxidase conjugated anti-rabbit antibody raised in goat (reference #7074P2: Cell Signaling), diluted 1 : 1000 in wash buffer containing 5% (w/v) skimmed milk powder. Finally, after another 3 x 10 min washes, the membranes were incubated for 1 minute with the chemiluminescent substrate ECL (GE Healthcare) and the emitted light was then detected in the Amersham Imager 600 luminescent image analyser (GE Healthcare). Images were analysed with ImageQuant TL software (GE Healthcare) to quantify the protein bands.

Example 1.16. Silencing with siRNA APOC2

Example 1.16.1. In vitro model: Same protocol that describes before in Example 1.11.

Example 1.16.1. Transfection: We used siRNAs against APO C2 and siRNAs control (ON- TARGETplus SMARTpool siRNA, L-010972-00-0005, and ON-TARGETplus non-targeting pool, 77D-001810-10-05, Cultek) to transfect VICs. LipofectamineTM RNAiMAX transfection reagent (Invitrogen) was used for siRNA delivery. Cells were transfected with 60 nM siRNA, using Lipofectamine RNAiMAX Reagent following the manufacturer’s instructions. After transfection (5h h), the medium was replaced, and cells were washed twice in culture medium and incubated for at least 8 h in in FIB of OM medium prior to Quantitative Real-Time PCR and calcification analysis. In calcification experiments and after transfection, VIC were incubated with an osteogenic medium for 7-10 days. When cells reached confluence, VICs were removed by trypsin-EDTA.

Example 1.16.1. Quantitative Real-Time PCR: Once VICs were removed by trypsin-EDTA, RNA extraction was performed using RNeasy Mini Kit (50974104, QIAGEN) based on the instructions provided by the manufacturer. Dnase I-treated total RNA (1 pg) was reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) with random hexamers. Primers with a concentration of 100 nanomoles were prepared (TABLE). To quantify gene expression, real-time PCR was performed using the qPCRBIO SyGreen Mix Hi-ROX SyGreen realtime PCR (K7PB20.12-05, PCR BIOSYSTEMS). As endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH, (Invitrogen) was used as endogenous control while aSMA, (Invitrogen) and TNFa, (Invitrogen) were used as markers of VICs activation. Each sample was amplified in duplicate. Data were normalized to GAPDH gene, and 2-ACt was used to calculate the expression of each gene. Similar results were obtained after normalization to either housekeeping gene. Quantitative RTPCR was carried out in a 7900HT Fast Real-Time PCR (Applied Biosystems).

APOC2_ F: 5'GTC AGC AAA GAC AGC CGC CCA 3' > SEQ ID NO: 1.

APOC2 R: 5 AGC CCC TCC ATC TTG GCC CTT 3' > SEQ ID NO: 2.

APOC2 _F: 5' GTT GGA GAC GAG GCT TAC CA 3' > SEQ ID NO: 3.

APOC2 R: 5' AGC CTC TGG AAT AGC TGG GA 3 ' > SEQ ID NO: 4.

GAPDH F: 5'GTC TCC TCT GAC TTC AAV AGC G 3 ' > SEQ ID NO: 5.

GADPH R: 5 ACC ACC CTG TTG CTG TAG CCA A 3' > SEQ ID NO: 6.

Alpha-SMA F: 5'CTA TGC CTC TGG ACG CCA AAC T 3' > SEQ ID NO: 7.

Alpha-SMA R: 5'CAG ATC CAG ACG CAT GAT GGC A 3 ' > SEQ ID NO: 8.

TNFa F: 5'CTC TTC TGC CTG CGT CCA TTT G 3' > SEQ ID NO: 9.

TNFa R: 5'ATG GGC TAC AGG CTT GTC ACT C 3' > SEQ ID NO: 10.

Example 1.16.1. Treatment with recombinant APOC2: VICs were exposed for 5 days APCO2 recombinant protein (ELB-PKSH032084, ElabScience) diluted in the corresponding cell culture media (FIB o OM) at 2 different concentrations: 10 ng/ml and 20ng/ml to analyse the effect of APOC2 in cell activation, calcification and lipid deposition. Example 1.16.1. Alizarin Red Staining: same protocol that describes before in Example 1.13. Alizarin Red Staining.

Example 1.16.1. Secreted calcium in cell culture media: The concentration of calcium in the culture media was determined using a commercial kit following the manufacturer's instructions (MAK022, Sigma-Aldrich; Merck KGaA). Briefly, a 5 mM (0.2 pg/pl) CalciumStandard Solution was produced by the addition of 10 pl of 500 mM Calcium Standard Solution to 990 pl water. The curve was generated from dilutions of this standard solution: 0 (assay blank), 0.4, 0.8, 1.2, 1.6 and 2.0 pg/well standards, respectively. Then, 50 pl of each culture media was used to test the calcium concentration. A total of 90 pl of chromogenic reagent was added to each well containing standards and was gently mixed. Subsequently, 60 pl of Calcium Assay Buffer (MAK022, Sigma- Aldrich) were added to each well and gently mixed. The cells were then incubated for 5-10 min at room temperature, and the plate was shielded from light during incubation. Absorbance at 575nm was measured and calcium concentration was calculated according to the standard curve.

Example 1.16.1. Oil Red Staining: The culture medium was removed from the wells, and the cells were washed. Cells were fixed were, then, fixed for 15 min with 4% paraformaldehyde and incubated with Oil Red O 0’6 % in 60% isopropanol for 30 min. VICs were washed with H2O four times to remove the dye. Finally, the stained VICs were observed by. Olympus 1X83 inverted microscope and analysed using Scan^AR software.

Example 2. Results

Example 2.1. APOC2 quantification and differential expression

For the quantitative analysis, the ratio of calcified: non-calcified tissue of each patient was compared. After the statistical analysis of the proteome and transcriptome of the AVs from patients with DAS, with or without DM, the results were compared to evaluate the correlation between them and to identify robust biomarkers for possible clinical use.

The APOC2 protein and APOC2 gene gave a -value < 0.05 and a fold change (FC) > 1.5 in both analyses, and these values moved in the same direction between the study groups (Figure 1).

Thus, the APOC2 protein was considered the most robust potential biomarker since it was identified in both the proteomic and transcriptomic analysis, and moreover, it was confirmed in plasma samples from an independent cohort of patients when studied by ELISA and turbidimetry (Figure 2). Using both these techniques, the amounts of this protein were higher in the group of diabetic patients relative to the non-diabetic patients (ELISA /?-value = 0.018; turbidimetry /?-value = 0.038).

Example 2.2. In vitro model

Once the VIC cell line was expanded, the cells were cultured for 7 days in FIB or OM medium. The cells were then stained with alizarin red and protein extracts were obtained to confirm osteogenic differentiation and to determine the onset of calcification. Enhanced alizarin red staining and more BMP -2 were found, with significant differences on day 7 in cells maintained in OM, confirming the onset of calcification (Figure 3).

Example 2.3. Plasma treatment of patients

Once the onset of calcification was determined, which appears to occur after 7 days (see above), the cells were exposed for 5 days to the plasma obtained from DAS patients with or without DM to analyse the APOC2 protein before onset of calcification. There was a trend towards a decrease in the APOC2 protein in cells treated with plasma from diabetic as opposed to nondiabetic patients (Figure 4).

Example 2.4. Treatment with recombinant APOC2 and silencing with siRNA APOC2

The cells were exposed to 10 and 20 ng/ml of recombinant APOC2. Calcification were more intense as concentration of APOC2 increased, both in FIB and MO. On the contrary, when APOC2 was silenced, calcification is less intense. In fact, when VICs were cultured in MO after the treatment with siRNA, levels of calcification were the same than in the FIB media. These results were confirmed also in the culture media. Where calcium levels were also measured (Figure 6).

Claims

1. APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use in a method for the treatment of aortic stenosis.

2. APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use, according to claim 1, in a method for the treatment of aortic stenosis in patients suffering from diabetes.

3. APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use, according to any of the previous claims, wherein the inhibitor inhibits the expression the APOC2 gene, or it carries out a pharmacological inhibition.

4. APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use, according to any of the previous claims, wherein the inhibitor is an RNA molecule preferably selected from the group consisting of a small interfering RNA molecule (siRNA), an anti-sense RNA molecule, a microRNA molecule (miRNA) and/or a short hairpin RNA (shRNA), and/or a genome editing tool preferably selected from the group consisting of zinc-finger proteins (ZFNs), transcription activator-like effector nucleases (TALENs) and/or CRISPR/Cas.

5. APOC2 inhibitor, or pharmaceutical composition comprising thereof, for use, according to any of the previous claims, wherein the inhibitor is a siRNA consisting of a nucleotide sequence complementary to the gene APOC2 and that interferes with the expression of the gene APOC2.

6. In vitro method for monitoring the efficacy of a treatment and/or for predicting the response to a treatment in a patient suffering from aortic stenosis which comprises: (a) Measuring the level of expression or activity of APOC2 in a biological sample isolated from the patient after the treatment; (b) wherein if the level of expression or activity of APOC2 determined in step (a) is statistically lower than the level of APOC2 before the treatment, it is indicative that the patient is responding to the treatment.

7. In vitro method for identifying and/or producing candidate compounds useful in the treatment of aortic stenosis which comprises: (a) Measuring the level of expression or activity of APOC2 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if the level of expression or activity of APOC2 determined in step (a) is statistically lower than the level of APOC2 determined before the administration of the candidate compound, is indicative that the candidate compound is effective in the treatment of aortic stenosis.

8. An in vitro method for the diagnosis and/or prognosis of aortic stenosis which comprises: (a) determining the level of expression of AP0C2 protein, or AP0C2 gene, in a biological sample obtained from a patient, b) wherein a deviation in expression from a pre-established reference value is indicative that the patient has aortic stenosis.

9. In vitro method, according to any of the previous claims, for the diagnosis and/or prognosis of aortic stenosis in patients suffering from diabetes.

10. In vitro method, according to any of the previous claims, wherein the biological sample is plasma, blood, serum or tissue from a heart biopsy.

11. In vitro method, according to any of the previous claims, comprising a) determining the level of expression of APOC2 protein, or APOC2 gene, in a biological sample of plasma, blood or serum obtained from a patient, b) wherein a level of expression above a previously established reference value is indicative that the patient has aortic stenosis, or that the patient with diabetes may develop aortic stenosis.

12. In vitro method, according to any of the previous claims, wherein the aortic stenosis is degenerative aortic stenosis.

13. In vitro method, according to any of the previous claims, wherein the diabetes is diabetes mellitus, preferably type 2 diabetes mellitus.

14. In vitro use of APOC2 protein, or the APOC2 gene, or of a kit comprising reagents for the measurement of APOC2 protein expression levels, or the APOC2 gene, for the diagnosis and/or prognosis of aortic stenosis, or to predict whether a patient may develop aortic stenosis.

15. In vitro use of APOC2 protein, or the APOC2 gene, or of a kit comprising reagents for the measurement of APOC2 protein expression levels, or the APOC2 gene, according to claim 14, for the diagnosis and/or prognosis of aortic stenosis in patients with diabetes, or to predict whether a patient with diabetes may develop aortic stenosis.