Compounds for use in the treatment of heart failure with preserved ejection fraction (HFPEF), through targeting of s100a9 and corresponding dimer partner and receptors

Abstract

The present invention refers to inhibitors of the S100 calcium- binding protein A9 (S100A9) / S100A8 – Toll Like Receptor 4(TLR4) dimer-receptor axis for use in the treatment of Heart Failure with preserved Ejection Fraction (HFpEF). The present invention further refers to pharmaceutical formulations comprising the said inhibitors. The present invention's use of compounds and pharmaceutical formulations may be advantageously employed in HFpEF by attenuating the deleterious effect on endothelial function and prevention of inflammation, production of reactive oxygen species, apoptosis, senescence and pro-fibrotic phenotype.

Description

COMPOUNDS FOR USE IN THE TREATMENT OF HEART FAILURE WITH PRESERVED EJECTION FRACTION (HFPEF), THROUGH TARGETING OF S100A9 AND CORRESPONDING DIMER PARTNER AND RECEPTORS Technical field of the invention The present invention relates to the technical field of human health; medical science; preparations for medical purposes; in particular, medicinal preparations containing active ingredients . State of the art Heart failure with preserved ejection fraction (HFpEF) prevalence is increasing at an alarming rate, especially among patients with a significant burden of cardiovascular comorbidity, including advanced age, diabetes mellitus, hypertension and obesity 1, 2. These comorbidities are believed to contribute to disease development and progression, at least in part, through the creation of a chronic low-grade systemic pro-inflammatory environment 3,4. In experimental models of HFpEF, systemic inflammation is associated with increased stiffness of myocardial interstitium and cardiomyocytes, as well as coronary microvascular dysfunction - two main hallmarks of clinical HFpEF 5, 6. In patients with HFpEF, circulating pro-inflammatory mediators associate with higher left ventricle filling pressure, disease severity and worse prognosis 4, 7. However, specific inflammatory pathways impacting the myocardium and contributing to HFpEF pathogenesis, remain largely elusive, yet their identification could be crucial for informing future therapeutic approaches. Extracellular vesicles (EVs) are important vehicles of paracrine and autocrine cell communication 8, playing a significant role in the development of several cardiovascular disorders (CVD) by driving critical pathological processes (e.g. inflammation and oxidative stress, etc) 9, 10. However, the role of EVs in the context of HFpEF has never been explored. Summary of the Invention The present invention originally discloses that circulating EVs deliver pathological signaling to cardiac endothelial cells contributing to the pathogenesis of HFpEF. Compared to a comorbidity matched control group (CTRL EVs), EVs isolated from a cohort of HFpEF patients (HFpEF EVs) potentiate cardiac endothelial cell activation, monocyte adhesion and senescence in vitro and endothelial dysfunction in ex vivo (as assessed by functional tests). Furthermore, endothelial cells stimulated by HFpEF EVs acquired a profibrotic phenotype, able to promote fibroblast-to-myofibroblast transition. Proteomic analysis of EVs, followed by functional validation in vitro and ex vivo further highlights the S100A9-TLR4 axis as main effector of HFpEF EVs deleterious effects. These findings support a novel disease mechanism in HFpEF by unveiling how EV-associated S100A9 can reach endothelial cells contributing to microvascular dysfunction and, subsequently, to cardiac fibrosis, two critical pathological mechanisms in HFpEF. S100A9 forms dimers with S100A8 to bind to TLR4, as such, the present invention refers to inhibitors of the S100 calcium-binding protein A9 (S100A9) / S100A8 - Toll Like Receptor 4(TLR4) dimer-receptor axis for use in the treatment of HFpEF, according to claim 1. In the present invention, the said inhibitors of the S100A9 / S100A8-TLR4 dimer-receptor axis comprise Resatorvid (TAK-242, CLI095); Eritoran; Dutasteride (DB01126); Buclizine (DB00354); Conivaptan (DB00872); Lomitapide (DB08827); Adapalene (DB00210); Lumacaftor (DB09280); Dihydroergotamine (DB00320); Bromocriptine (DB01200); Tolvaptan (DB06212) Nandrolone phenpropionate (DB00984); Flunarizine (DB04841); Irinotecan (DB00762); Dihydrotachysterol (DB01070); Antrafenine (DB01419); Nilotinib (DB04868); Medrogestone (DB09124); Itraconazole (DB01167) ; Doxercalciferol (DB06410); Ponatinib (DB08901); Netupitant (DB09048); Difenoxin (DB01501) ; Paricalcitol (DB00910); Pimozide (DB01100); Tasosartan (DB01349) ; Alfacalcidol (DB01436); Fluspirilene (DB04842); Azilsartan medoxomil (DB08822); Cabozantinib (DB08875); Lorpiprazole (DB09195) Ergocalciferol (DB00153); Loperamide (DB00836); Drospirenone (DB01395) Pranlukast (DB01411); Ziprasidone (DB00246); Meclizine (DB00737); Sertindole (DB06144); Lurasidone (DB08815); Vorapaxar (DB09030); Umeclidinium (DB09076), as well as S100A9 Inhibitors, such as ABR-238901; Tasquinimod (ABR-215050); Paquinimod (ABR 25757); laquinimod (ABR-215062); Roquinimex (ABR212626); Azeliragon; FPS-ZM1; RAGE 229; AC-73; antagonist peptide-9 (AP9) and combinations thereof, according to claim 2. The present invention further refers to a pharmaceutical formulation for use for use in the treatment of HFpEF, comprising inhibitors of the S100A9 / S100A8-TLR4 dimerreceptor axis, according to claim 3. In another embodiment, in the pharmaceutical formulation mentioned above, the said inhibitors comprise Resatorvid (TAK-242, CLI095); Eritoran; Dutasteride (DB01126); Buclizine (DB00354); Conivaptan (DB00872); Lomitapide (DB08827); Adapalene (DB00210); Lumacaftor (DB09280); Dihydroergotamine (DB00320); Bromocriptine (DB01200); Tolvaptan (DB06212) Nandrolone phenpropionate (DB00984); Flunarizine (DB04841); Irinotecan (DB00762); Dihydrotachysterol (DB01070); Antrafenine (DB01419); Nilotinib (DB04868); Medrogestone (DB09124); Itraconazole (DB01167) ; Doxercalciferol (DB06410); Ponatinib (DB08901); Netupitant (DB09048); Difenoxin (DB01501)  ;  Paricalcitol (DB00910); Pimozide (DB01100); Tasosartan (DB01349)   ; Alfacalcidol (DB01436); Fluspirilene (DB04842); Azilsartan medoxomil (DB08822); Cabozantinib (DB08875); Lorpiprazole (DB09195) Ergocalciferol (DB00153); Loperamide (DB00836); Drospirenone (DB01395) Pranlukast (DB01411); Ziprasidone (DB00246); Meclizine (DB00737); Sertindole (DB06144); Lurasidone (DB08815); Vorapaxar (DB09030); Umeclidinium (DB09076), as well as S100A9 Inhibitors, such as ABR-238901; Tasquinimod (ABR-215050); Paquinimod (ABR 25757); laquinimod (ABR-215062) ; Roquinimex (ABR212626); Azeliragon; FPS-ZM1; RAGE 229; AC-73; antagonist peptide-9 (AP9) and combinations thereof, according to claim 4. Detailed description of the Invention EVs are isolated from plasma of HFpEF (HFpEF EVs) and from comorbidity-matched control patients without clinical, imaging and analytical evidence of HF (CTRL EVs) by size exclusion chromatography (SEC). Dynamic light scattering (DLS) analysis shows the fractions enriched in particles with a more homogeneous size compatible with small EVs (50 to 150 nm of diameter). Immunoblotting for EV markers CD63 and CD81 and bead-assisted flow cytometry for CD71 and CD9 confirms fractions in which EVs are more abundant. EVs are then concentrated by ultrafiltration and characterized according to MISEV2023 guidelines. Considering a cut-off size of 50-150 nm, a similar concentration of EVs is found between CTRL and HFpEF patient groups. Challenging Microvascular endothelial cells (MVECs) with EVs (1x108 EVs / mL) collected from the two patient groups shows HFpEF EVs induce higher levels of MVECs activation, as demonstrated by increased percentage of ICAM1+ and VCAM1+ cells, (Fig. 1A) and higher monocyte (THP1) adhesion (Fig. IB), compared to CTRL EVs. Furthermore, HFpEF EVs induce increased production of reactive oxygen species (ROS) (Fig. IC) and increased intracellular levels of cleaved Caspase 3 (C-caspase 3) on MVECS. Collectively, HFpEF EVs show enhanced capacity to promote endothelial inflammation, oxidative stress and apoptosis. Senescence contributes to HFpEF development in experimental animals and correlates with disease severity in HFpEF patients. Comparing with cells treated with CTRL EVs, MVECs stimulated with HFpEF EVs showed increased activity of beta galactosidase (SA-p-Gal), a biomarker of cellular senescence (Fig. ID) . The senescent phenotype is confirmed by additional hallmarks, namely a significant increase on: i) cell size of SA-p-Gal+ MVECs (Fig. IE); ii) levels of DNA-damage, as shown by increased frequency of cells with phosphorylated histone variant H2AX (pH2AX) (Fig. IF); and iii) expression of cell cycle arrest associated genes such as CDNK1A (encodes p21) and CDNK2A (encodes pl6) (Fig. 1G). Moreover, the primary pathway responsible for triggering the emergence of senescence-associated secretory phenotype (SASP), NF-kB15, is activated in MVECs exposed to HFpEF EVs, as shown by increased nuclear intensity of NF-kB (Fig. 1H). Considering that senescent endothelial cells contribute to tissue fibrosis by secretion of profibrotic mediators and pro-inflammatory cytokines, the impact of EV-stimulated MVECs on cardiac fibroblast-to-myofibroblast differentiation is assessed. For this purpose, conditioned media (MVEC-CM) is collected from MVECs exposed to EVs from the two patient groups, and used to challenge human cardiac fibroblasts (hCF) in vitro. Consistently, an increase in alpha-smooth muscle actin+ (a-SMA+)-expressing cells and higher deposition of collagen type 1, is observed in hCF treated with MVEC-CM following stimulation with HFpEF EVs (Fig. 1I-J). Taken together, these results disclose that circulating EVs from HFpEF patients have an enhanced capability to induce a senescent phenotype on endothelial cells, which in turn become pro-fibrotic. The biological effect of HFpEF EVs translates into functional impact. Rat mesenteric arteries (MA) are challenged with HFpEF and CTRL EVs and the response to the endotheliumdependent vasodilator acetylcholine (ACh), the endotheliumindependent vasodilator sodium nitroprusside (SNR) and the vasoconstrictor U46619 (U4), are studied by wire myography (Fig. 2A) . MA exposed to HFpEF EVs exhibit no significant differences in the contractile response to U4 compared to CTRL EVs (Fig. 2B) . Surprisingly, relaxation of MA in response to ACh and SNP is significantly impaired in HFpEF EVs when compared to the CTRL group (Fig. 2C-D) . Of note, the area under the curve in response to ACh, which is a representation of the relaxation efficiency, correlates with important parameters of renal dysfunction, plasma creatinine levels and estimated glomerular filtration rate in HFpEF patients (Fig. 2E-F). Thus, circulating EVs are drivers of endothelium-dependent vasodilation impairment in HFpEF. To obtain mechanistic insights on the EV-mediated effects, proteomic analysis is performed in HFpEF EVs and CTRL EVs by liquid chromatography-tandem mass spectrometry (nanoLC-MS / MS) and the analysis is focused on proteins detected in over 90% of the samples. Considering the comparison between the two experimental groups, unsupervised clustering shows considerable discrimination between samples from the two patient cohorts (Fig. 3A) and overall, 34 proteins show differential abundance (p<0.05) between CTRL and HFpEF EVs, 8 of which being more abundant in HFpEF EVs, whereas 26 are enriched in CTRL EVs (Fig. 3B) . The most significant enriched biological processes related with upregulated proteins in HFpEF EVs is Leukocyte Aggregation and Brown Fat Cell Differentiation, both associated with S100 calcium binding protein A9 (S100A9) and adiponectin (ADIPOQ) , respectively (Fig. 3C) . Proteins decreased in HFpEF EVs are associated with Reverse Cholesterol Transport and Regulation of Opsonization (Fig. 3D) . To refine target selection, a VIP (Variable Importance in Projection) score plot is performed to highlight proteins more relevant to each group (Fig. 3E) . S100A9 (migration inhibitory factor-related protein 14 (MRP14) or calgranulin B) is the protein that presents the highest predicted relevance for the HFpEF group. An ELISAbased quantification is performed in intact EVs (Non-Lysed) and after treatment with RIPA(Radioimmunoprecipitation assay) (Lysed) buffer to disrupt EV membranes. This protein detected exclusively after EV lysis (Fig. 3F), support that S100A9 is carried inside EVs. The present invention thus discloses a causal association between the deleterious effect promoted by HFpEF-EVs in MVECs and the S100A9 and corresponding signaling pathway mediated by toll-like receptor 4 (TLR4). Both direct inhibition of S100A9 (by paquinimod - "Paq") and of TLR4 signaling pathway (using CLI095 - herein abbreviated by "Cli") decreased monocyte adhesion (Fig. 4A) and the frequency of ICAM1+ and VCAM1+ MVECs following stimulation with HFpEF EVs, but not CTRL EVs (Fig. 4B) . Moreover, the increase in ROS and activation of NFkB pathway in MVECs exposed to HFpEF EVs is counteracted (Fig. 4C-D). In addition, inhibition reduces HFpEF EVs-induced MVEC senescence (Fig. 4E-F) . Of note, hCF activation induced by the secretome of MVECs exposed to HFpEF EVs is reverted with S100A9 inhibition whereas no effect is observed with TLR4 inhibition (Fig. 4G) . Surprisingly, both Paq and Cli reverts HFpEF-EVs-induced mesenteric endothelial dysfunction, as observed by an increase in endothelium-dependent relaxation induced by acetylcholine (ACh) (Fig. 5A). In terms of endothelial-independent relaxation induced by SNP (Fig. 5B), Pag reverted the delay in relaxation induced by HFpEF EVs, whilst no differences were found regarding SNP when MA were exposed to Cli (Fig. 5B) . Overall, the present invention reveals that high levels of S100A9 in HFpEF patients are responsible for the harmful effects of HFpEF EVs on endothelial cells and discloses inhibitors of the S100A9 / S100A8-TLR4 dimer-receptor axis for use in the treatment of HFpEF. In other embodiments, the said inhibitors comprise Resatorvid (TAK-242, CLI095); Eritoran; Dutasteride (DB01126); Buclizine (DB00354); Conivaptan (DB00872); Lomitapide (DB08827); Adapalene (DB00210); Lumacaftor (DB09280); Dihydroergotamine (DB00320); Bromocriptine (DB01200); Tolvaptan (DB06212) Nandrolone phenpropionate (DB00984); Flunarizine (DB04841); Irinotecan (DB00762); Dihydrotachysterol (DB01070); Antrafenine (DB01419); Nilotinib (DB04868); Medrogestone (DB09124); Itraconazole (DB01167) ; Doxercalciferol (DB06410); Ponatinib (DB08901); Netupitant (DB09048); Difenoxin (DB01501) ; Paricalcitol (DB00910); Pimozide (DB01100); Tasosartan (DB01349) ; Alfacalcidol (DB01436); Fluspirilene (DB04842); Azilsartan medoxomil (DB08822); Cabozantinib (DB08875); Lorpiprazole (DB09195) Ergocalciferol (DB00153); Loperamide (DB00836); Drospirenone (DB01395) Pranlukast (DB01411); Ziprasidone (DB00246); Meclizine (DB00737); Sertindole (DB06144); Lurasidone (DB08815); Vorapaxar (DB09030); Umeclidinium (DB09076), as well as S100A9 Inhibitors, such as ABR-238901; Tasquinimod (ABR-215050); Paquinimod (ABR 25757); laquinimod (ABR-215062); Roquinimex (ABR212626); Azeliragon; FPS-ZM1; RAGE 229; AC-73; antagonist peptide-9 (AP9) and combinations thereof . Another embodiment of the present invention refers to a pharmaceutical formulation comprising the above mentioned inhibitors, alone or in combination, for use in the treatment of HFpEF. Brief description of the Figures Figure 1. HFpEF EVs promote endothelial inflammation and the acquisition of a senescent and profibrotic phenotype. A. Percentage of ICAM1+ and VCAM1+ human cardiac microvascular endothelial cells (MVECs) after incubation with HFpEF and CTRL-derived EVs (n>10 / group). B. Representative image and respective quantification of THP-1 monocytes (green) adhered to MVECs (magenta) stimulated with EVs from HFpEF and CTRL patients (nuclei counterstained with DAPI, blue) (n>15 / group). C. Relative levels of reactive oxygen species (ROS) in MVECs (n>15 / group). D. Representative image and respective quantification of SA-p-Gal+ MVECs (n>15 / group). E. Cell size of SA-p-Gal+ and non-SA-p-Gal MVECs (n >30 / group). F. Representative image and respective quantification of pH2AX+ MVECs (with at least 7 foci per nuclei) (n>14 / group). G. Gene expression of target cell cycle inhibitors in MVECs (n>15 / group). Values were normalized to MVECs. H. Representative image of NFkB immunostaining and respective quantification of MVECs with nuclear NFkB (n>9 / group). I and J. Representative images and respective quantification of a-SMA+ cells (I.) and area of collagen type I (Collagen-1) deposition (J.) in hCF exposed to CM produced by MVECs in response to HFpEF and CTRL EVs(n>l6 / group). Scale bars: 40pm. For human EVs isolation and concentration, peripheral blood samples were obtained from HFpEF and comorbidity-matched control patients at Centro Hospitalar Universitario Sao Joao (Porto, Portugal) included in the NETDiamond (NEw Targets in DIAstolic heart failure: from coMOrbidities to persoNalizeD medicine) study. The study was approved by local Ethics Committee and met the ethical standards of the Declaration of Helsinki and donors signed an informed consent according to the Portuguese legislation. Within two hours after blood collection, using EDTA as an anti-coagulant, plasma was separated from the buffy coat through centrifugation at 12000 x g, 30 minutes (min) at room temperature (RT) , without brake and acceleration. Then plasma was centrifuged at 2500 x g for 30 minutes and stored at -80°C. Extracellular vesicles (EVs) were isolated and concentrated by size exclusion chromatography (SEC) in sepharose CL2B300 (GE Healthcare). All samples were then stored at -80 °C. To study effect of EVs on cardiac cells through culture of MVECs with EVs, Human cardiac MVECs microvascular endothelial cells (MVECs; #70130, Lonza) were seeded at a density of 20000 cells / cm2 in EGM. After 24 h, MVECs were washed with PBS and left in the following conditions: 1) EGM without FBS supplemented with 1x108 particles / mL as reported in of EVs from HFpEF or CTRL patients; 2) EGM without FBS supplemented with 1x108 particles / mL of EVs from HFpEF or CTRL patients and or without CLI095 (Invivogen, 10 pM) or Paquinimod (Sigma Aldrich, 15 pM) . MVECs were incubated for 48 h at 37 °C, 5% CO2, after which cells were collected for subsequent analysis. To collect conditioned media (MVEC-CM), MVECs were washed with PBS and incubated in EGM without FBS for an additional period of 24 h, at 37 °C, 5% CO2. MVEC-CM was collected and centrifuged at 2500 x g, 30 min, at 4 °C, and stored at - 80 °C until further use. To culture of hCF with CM-MVECs, adult cardiac fibroblasts (HCF, Cell Applications, Inc.) were seeded at a density of 36500 cells / cm2 in Dulbecco's Modified Eagle's Medium (DMEM; Thermofisher) supplemented with 10% FBS (Lonza), 1% Penicillin-Streptomycin (P / S; Biowest) and 0.2 mM 2-phospho-Lascorbic acid (Asc-2P; Sigma Aldrich, Co.). After 24 h, the media was changed to working media (WM) consisting of DMEM high glucose with 0.1% FBS + 0.2 mM Asc. Acid + 1% P / S. After two days, the media was changed to WM containing 10% (V / V) of CM-MVEC and cells were incubated at 37 °C, 5% C02. The renewal with CM-MVEC media was performed every two days. At day 7 post CM-MVECs supplementation, HCF were fixed and processed for further analysis. To perform the THP1 adhesion assay, MVECs were stimulated with EVs for 24 h as described above. After 24 h, MVECs were washed with PBS and incubated with CellMask Deep Red Plasma Membrane stain (Invitrogen, 1:1000) and Hoechst dye (Sigma, 1:500) for 30 min at 37 °C. At the same time, THP1 cells were incubated with calcein (Sigma, 1:500) for 30 min, 37 °C. Stepwise, MVECs and THP1 were washed twice with PBS and 53000 THPl / cm2 were added to MVECs and left to adhere for 30 min at 37 °C. Then, MVECs were washed at least three time to remove the non-adherent THP1 cells and images were immediately acquired on Opera Phenix Plus (Perkin Elmer). (Revvity). Data was analyzed using Harmony software (Revvity). For ROS detection, MVECs were challenged with EVs as reported above. After 48h, the media was changed for Krebs medium supplemented with 0.01 pM CaC12 and ROS probe (H2DCFDA, lOpM, Invitrogen) for 30 min, at 37°C, 5% CO2. Then, this media was replaced with Krebs media supplemented with 0.01 pM of CaC12. The absorbance was monitored during 30 min, at an excitation of 480 nm and emission of 550 nm, with a cut off of 530 nm (Synergy). ROS levels were obtained by calculating the slope of the line. For immunofluorescence, cardiac cells were fixed with PFA 4% in PBS pH 7.4 for 12 min, at RT, and washed with PBS (3x, 5 min, RT). Next, the cells were permeabilized with Triton X-100 0.1 to 1% in PBS for 5 min, at RT. Stepwise, cells were incubated with blocking solution (4% FBS, 1% BSA in PBS) for Ih at RT. Cells were subsequently incubated with primary antibody in blocking solution, overnight at 4°C. In the following day, cells were washed with PBS (3x, 5 min, RT) and incubated with secondary antibody in blocking solution for Ih at RT, protected from light. Following this incubation, cells were washed with PBS (3x, 5min, RT) and incubated with DAPI 1:3000 and when necessary with cell mask green (Invitrogen, 1: 1000) in PBS for 30 min, at RT, protected from light. Cells were washed with PBS (3x, 5 min, RT), and images were acquired on Operetta CLS. For the SA-p-gal assay in MVECs, after incubation with EVs, MVECs were incubated for 90min in EGM with 100 nM Bafilomycin Al (Sigma-Aldrich). Fluorescein di-p-D-galactopyranoside (33pM, Sigma-Aldrich) was then added to the medium during 90min at 37°C. Cells were fixed in 4% paraformaldehyde for 15min, rinsed with PBS, and permeabilized with 0.1% Triton-X100 in PBS for 15min. Finally, cells were counterstained with Ipg / ml DAPI (Sigma-Aldrich) and immediately, images were acquired on Operetta CLS. Images were analyzed using Fiji software. Figure 2. EVs from the plasma of HFpEF patients impair vascular relaxation. A. Rat mesenteric arteries were exposed to EVs from CTRL and HFpEF patients for 24 hours and wire myography was performed. B. Contractile response to U-46619 (U4) (IpM) . Each dot represents the response of a single artery (~2 arterial rings per patient) (n=21 / CTRL; n=35 / HFpEF) . C. Dose response curves and area under the curve (AUG) to the endothelium-dependent vasodilator acetylcholine (ACh) in MA pre-contracted with U4 (n=21 / CTRL, n=35 / HFpEF). D. Dose response curves and AUG to the endotheliumindependent vasodilator sodium-nitroprusside (SNP) in MA pre-contracted with U4 (n=13 / CTRL, n=27 / HFpEF) . E. and D. Association between AUG of the endotheliumdependent vasodilator ACh with the levels of the plasma creatinine (E.) and estimated glomerular filtration rate (eGFR) (F.) of HFpEF patients. Values are presented as mean ± SEM. Regarding animal model and vessel dissection, all experimental procedures were approved by the Research Ethical Committee of Universidad Complutense de Madrid and by the Madrid regional authorities (ref. PROEX 334.8 / 23). The study was conducted in accordance with the National Institute of Health (NIH), Guide for the Care and Use of Laboratory Animals and the Spanish Policy for Animal Protection RD 53 / 2013, which meets the European Union Directive 2010 / 63 / UE on the protection of animals used for experimental and other scientific purposes. Male and female 11 weeks old Wistar Han rats were used for the experiment. Animals were sacrificed by C02 inhalation. Mesentery vascular bed was isolated from each rat and bathed in krebs solution. Mesenteric arteries (MA) were dissected to remove the fat tissue, the connective tissue and without provoking any stretching. The arteries were cut into 2 mm-long segments. For culture of mesenteric arteries with EVs, MA were incubated in DMEM supplemented with glucose (4.5 g / L), nonessential amino acid solution (lx), penicillin (100 U / mL), streptomycin (0.1 mg / mL), and amphotericin B (0.25 pg / mL) with the following stimuli: 1) 1x108 particles / mL of EVs from HFpEF or CTRL patients; 2) 1x108 particles / mL of EVs from HFpEF or CTRL patients and CLI095 (Invivogen, 0.5 pM) or paquinimod (Sigma Aldrich, 0.125 Mm). MA were incubated for 20 to 24 h at 37°C, 5% CO2. Figure 3. Proteomic analysis of circulating EVs from HFpEF and CTRL patients. A. Principal component analysis discriminating the samples from each patient group. B. Unsupervised heatmap of the relative amount of altered proteins in CTRL and HFpEF EVs. C. and D. GO analysis of Biological Processes associated with proteins upregulated in HFpEF EVs (C.) and CTRL EVs (D.). E. Variable importance in projection (VIP) scores of important components identified by PLS-DA (Partial Least Squares - Discriminant Analysis) F. Quantification of S100A9 in HFpEF EVs diluted in PBS (nonLysed) or lysed in RIPA (Lysed) (n=4 / group) . For proteomic analysis of EVs, 10 pg EVs were reduced with dithiothreitol at 37°C for 60 minutes and then alkylated with iodoacetamide at 25°C for 30 minutes. The protein extract was diluted with 200 mM ammonium bicarbonate, digested with the endoproteinase LysC at 37°C for 6 hours, further diluted and digested overnight with trypsin. The peptide mixture was acidified, desalted with a MicroSpin C18 column and analyzed with an LTQ-Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1200 system. Bovine serum albumin was used as a stability control and QCloud monitored the performance of the instrument. For proteomic data analysis and S100A9 validation, the LC-MS / MS raw files were processed using MaxQuant, where proteins were identified using the UniProtKB Homo sapiens proteome with an FDR of 1%. Quantification was performed using the MaxLFQ algorithm. Results were analyzed in Perseus and MetaboAnalyst 5.0, with a significance threshold of p < 0.05 for t-tests. PCA, multi-stream plots, histograms and volcano plots were generated, and STRING analyzed the biological functions of the protein clusters. S100A9 levels were quantified in EVs resuspended in PBS and following RIPA treatment using Luminex Discovery Assay (Biotechne, RD systems) accordingly with manufacture instructions. Figure 4. Inhibition of S100A9-TLR4 pathway rescues HFpEF EV-induced endothelial alterations. MVECs were exposed to HFpEF EVs in the presence or absence of the S100A9 inhibitor Paquinimod (Paq) or the TLR4 inhibitor CLI095 (CLI). A. Representative image and respective quantification of THP-1 monocytes (green) adhered to MVECs (magenta) (nuclei counterstained with DAPI, blue) (n>7 / group). B. Percentage of ICAM1+ and VCAM1+ MVECs assessed by flow cytometry (n>7 / group) . C. Relative levels of ROS in MVECs (n>7 / group) . D. Representative images of NFkB immunofluorescence and respective quantification of MVECs with nuclear localization of NFkB (n>10 / group) . E. Percentage of SA-p-Gal+ MVECs (n=6 / group). F. Representative mages and respective quantification of of yH2AX foci (magenta) in MVECs. G. Representative images and respective quantification of a-SMA+ hCF exposed to CM from MVECs previously stimulated with HFpEV and CTRL EVs, in the presence or absence of Paq and CLI (n>5 / group). Scale bar, 4 0pm. Figure 5. Inhibition of S100A9-TLR4 pathway attenuates HFpEF EVs-induced endothelial dysfunction. Rat mesenteric arteries were exposed to EVs from HFpEF patients, in the presence or absence of the S100A9 inhibitor Paq or the TLR4 inhibitor CLI, and subjected to wire myography. A. Dose response curves and AUG to the endothelium-dependent vasodilator ACh in MA pre-contracted with U4 (n>20 / group). B. Dose response curves to the endothelium-independent vasodilator SNP (n>18 / group) in MA pre-contracted with U4 (n>12 / group). Values are presented as mean ± SEM. Overall, in terms of statistical analysis, statistical testing was performed using GraphPad® Prism 8.0 Software. Shapiro normality test was used to evaluate normal distribution of data. Normally distributed data was tested with independent sample Student's t test and one-way ANOVA (Bonferroni's post hoc test) for two or three groups, respectively. Outliers were excluded by ROUT analysis. Nonnormally distributed data was tested with Mann-Whitney U test and Kruskal-Wallis one-way ANOVA for two or three groups, respectively. Data with two independent variables were compared with two-way ANOVA followed by pairwise comparison using Bonferroni's posthoc test. Associations between AUG ACh, eGFR and plasma creatinine were performed using Person r correlation. Results are presented as column bars with Mean ± SEM. Differences between groups were considered significant when p<0.05. References 1. Shah SJ, Katz DH and Deo RC. Phenotypic spectrum of heart failure with preserved ejection fraction. Heart Fail Clin. 2014;10:407-418. 2. Dunlay SM, Roger VL and Redfield MM. Epidemiology of heart failure with preserved ejection fraction. Nature reviews Cardiology. 2017;14:591-602. 3. Ferrucci L and Fabbri E. Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty. Nature reviews Cardiology. 2018;15:505-522. 4. Wijk SS-v, Tromp J, Beussink-Nelson L, Hage C, Svedlund S, Saraste A, Swat SA, Sanchez C, Njoroge J, Tan R-S, Fermer ML, Gan L-M, Lund LH, Lam CSP and Shah SJ. Proteomic Evaluation of the Comorbidityinflammation Paradigm in Heart Failure With Preserved Ejection Fraction. Circulation. 2020;142:2029-2044. 5. Paulus Walter J and Tschdpe C. A Novel Paradigm for Heart Failure With Preserved Ejection Fraction. Journal of the American College of Cardiology. 2013;62:263-271. 6. Hamdani N, Franssen C, Lourengo A, Falcao-Pires I, Fontoura D, Leite S, Plettig L, Lopez B, Ottenheijm CA, Becher PM, Gonzalez A, Tschdpe C, Diez J, Linke WA, Leite- Moreira AF and Paulus WJ. Myocardial titin hypophosphorylation importantly contributes to heart failure with preserved ejection fraction in a rat metabolic risk model. Circulation Heart failure. 2013;6:1239-49. 7. Hage C, Michaelsson E, Linde C, Donal E, Daubert J-C, Gan L-M and Lund LH. Inflammatory Biomarkers Predict Heart Failure Severity and Prognosis in Patients With Heart Failure With Preserved Ejection Fraction. Circulation: Cardiovascular Genetics. 2017;10:eOOl633. 8. Tetta C, Ghigo E, Silengo L, Deregibus MC and Camussi G. Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine. 2013;44:11-9. 9. Bang C, Batkai S, Dangwal S, Gupta SK, Foinquinos A, Holzmann A, Just A, Remke J, Zimmer K, Zeug A, Ponimaskin E, Schmiedl A, Yin X, Mayr M, Halder R, Fischer A, Engelhardt S, Wei Y, Schober A, Fiedler J and Thum T. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J Clin Invest. 2014;124:2136-46. 10. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A and Mallat Z. Circulating Microparticles From Patients With Myocardial Infarction Cause Endothelial Dysfunction. Circulation. 2001,-104 :2649-2652 . Lisbon, 6th December 2024

Claims

1. Inhibitors of the S100 calcium-binding protein A9 (S100A9) / S100A8 - Toll Like Receptor 4(TLR4) dimer-receptoraxis for use in the treatment ejection fraction (HFpEF) deleterious effect of EVs prevention of a pro-fibroticof heart failure with preserved, through attenuating the on endothelial function andphenotype .

2. Use according to claim 1 wherein the said inhibitors compriseResatorvid   (TAK-242,   CLI095);   Eritoran; Dutasteride(DB01126);   Buclizine   (DB00354);   Conivaptan (DB00872);Lomitapide   (DB08827);   Adapalene   (DB00210); Lumacaftor(DB09280);   Dihydroergotamine (DB00320); Bromocriptine(DB01200);  Tolvaptan (DB06212) Nandrolone phenpropionate(DB00984);  Flunarizine (DB04841); Irinotecan (DB00762);Dihydrotachysterol    (DB01070);    Antrafenine    (DB01419);Nilotinib  (DB04868);  Medrogestone  (DB09124);  Itraconazole(DB01167) ; Doxercalciferol (DB06410); Ponatinib (DB08901);Netupitant  (DB09048); Difenoxin  (DB01501) ;  Paricalcitol(DB00910);   Pimozide (DB01100); Tasosartan (DB01349)   ;Alfacalcidol (DB01436); Fluspirilene (DB04842); Azilsartan medoxomil  (DB08822);  Cabozantinib  (DB08875);  Lorpiprazole(DB09195) Ergocalciferol (DB00153); Loperamide (DB00836); Drospirenone (DB01395) Pranlukast (DB01411); Ziprasidone (DB00246); Meclizine (DB00737); Sertindole (DB06144); Lurasidone (DB08815); Vorapaxar (DB09030); Umeclidinium (DB09076), as well as S100A9 Inhibitors, such as ABR-238901; Tasquinimod (ABR-215050); Paquinimod (ABR 25757); laquinimod (ABR-215062); Roquinimex (ABR212626); Azeliragon; FPS-ZM1; RAGE 229; AC-73; antagonist peptide-9 (AP9) and combinations thereof .

3. Pharmaceutical formulation for use for use in the treatment of HFpEF characterized by comprising inhibitors of the S100A9 / S100A8-TLR4 dimer-receptor axis.WO 2025 / 122021                                   PCT / PT2024 / 0500474. Pharmaceutical formulation according to claim 3 wherein the said inhibitors comprise Resatorvid (TAK-242,  CLI095);Eritoran; Dutasteride (DB01126); Buclizine (DB00354);Conivaptan (DB00872); Lomitapide (DB08827); Adapalene (DB00210); Lumacaftor (DB09280); Dihydroergotamine (DB00320); Bromocriptine (DB01200); Tolvaptan (DB06212)Nandrolone phenpropionate (DB00984); Flunarizine (DB04841); Irinotecan (DB00762); Dihydrotachysterol (DB01070); Antrafenine (DB01419); Nilotinib (DB04868); Medrogestone (DB09124); Itraconazole (DB01167)    ; Doxercalciferol(DB06410); Ponatinib (DB08901); Netupitant (DB09048); Difenoxin (DB01501)   ; Paricalcitol (DB00910); Pimozide(DB01100); Tasosartan (DB01349)  ; Alfacalcidol (DB01436);Fluspirilene (DB04842); Azilsartan medoxomil (DB08822);Cabozantinib (DB08875); Lorpiprazole (DB09195) Ergocalciferol (DB00153); Loperamide (DB00836); Drospirenone (DB01395) Pranlukast (DB01411); Ziprasidone (DB00246);Meclizine (DB00737); Sertindole (DB06144); Lurasidone (DB08815); Vorapaxar (DB09030); Umeclidinium (DB09076); ABR-238901; Tasquinimod (ABR-215050); Paquinimod (ABR 25757); laquinimod (ABR-215062); Roquinimex (ABR212626), Azeliragon; FPS-ZM1; RAGE 229; AC-73; antagonist peptide-9 (AP9) and combinations thereof.Lisbon, 6th December 2024

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