Composition comprising bindarit for preventing or treating heart disease

Abstract

The present invention relates to a composition comprising bindarit for preventing, alleviating or treating heart disease. The composition promotes survival and proliferation of endothelial cells and regulates cell stress and apoptosis, and thus has an effect of preventing, alleviating or treating heart disease and alleviating cardiac dysfunction caused by the disease.

Description

A composition for the prevention or treatment of heart disease containing vindarit.

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of heart diseases, including myocardial infarction, comprising Bindarit, or to the use of Bindarit for the protection of endothelial cells.

[0002] Myocardial infarction triggers left ventricular (LV) remodeling and is one of the leading causes of death worldwide. Myocardial infarction is generally caused by the rupture of coronary artery plaques, which can occur when oxygen supply to the myocardium is insufficient due to myocardial cell death, acute inflammation, angiogenesis, and fibrosis formation. Endothelial cells play a pivotal role in restoring cardiac function after myocardial infarction through vascular recanalization and improved perfusion of the infarct site. Furthermore, endothelial cells contribute to tissue repair by aggregating recovery cells and regulating the remodeling of the extracellular matrix, which is crucial for inflammation and cardiac function recovery. Reducing the size of the infarction by enhancing vascular regeneration is the most effective response to myocardial infarction, and angiogenesis is the primary mechanism for regenerating blood vessels in the infarcted heart. Recently, it has been revealed that stimulation of the proliferative and motile phenotypes of endothelial cells is essential for angiogenesis. Incomplete regeneration of the infarct site can lead to the accumulation of fibrous tissue, reduced barrier function, and microvascular dysfunction associated with permeability and inflammation, ultimately resulting in impaired angiogenesis. Consequently, significant efforts are currently focused on identifying endothelial cells that induce vascular homeostasis and angiogenesis in the infarct site, as well as elucidating their regulatory mechanisms.

[0003] Furthermore, endoplasmic reticulum (ER) stress signaling plays a crucial role in regulating cell proliferation and death in response to cellular stress and is associated with cardiovascular homeostasis and pathogenesis. Ischemic injury, such as myocardial infarction, disrupts the regulatory functions of the ER, activating unfolded protein responses (UPRs) mediated through specific pathways. Phosphorylation of eukaryotic initiation factor 2-alpha (eIF2α) by PRKR-like ER kinases (PERKs) induces overall inhibition of translation and promotes the translation of distinct proteins, such as the activation of transcription factor 4 (ATF4), which regulates genes involved in protein folding, oxidative stress, and amino acid metabolism. Under prolonged ER stress conditions, ATF4-mediated expression of the CCAAT / enhancer-binding homolog (CHOP) initiates apoptotic pathways. Several studies have reported the role of ER stress in angiogenesis and EC dysfunction through the regulation of cell proliferation and apoptosis. In contrast to EC apoptosis induction caused by uncontrolled or prolonged ER stress, vascular endothelial growth factor (VEGF) signaling contributes to angiogenesis by activating cell survival signals through transient ER stress and high levels of AKT phosphorylation activation. However, the detailed molecular mechanisms that balance the destruction and proliferation of ECs in response to ischemic injury have not been fully elucidated.

[0004] The inventors have discovered that Bindarit plays an important role in the survival and proliferation of endothelial cells (ECs) and can have a preventive or therapeutic effect against heart diseases such as myocardial infarction or ischemic cardiomyopathy by protecting endothelial cells from cell death induced by myocardial cell death, acute inflammation, angiogenesis, and fibrosis formation, and have completed the present invention.

[0005] Therefore, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of heart disease comprising vindarite or a pharmaceutically acceptable salt thereof.

[0006] Another objective of the present invention is to provide a health functional food composition for the prevention or improvement of heart disease comprising bindarite or a food-grade acceptable salt thereof.

[0007] Another objective of the present invention is to provide a composition for protecting endothelial cells (EC) comprising vindarit.

[0008] To achieve the above objectives, the present invention provides a pharmaceutical composition for the prevention or treatment of heart disease comprising vindarite or a pharmaceutically acceptable salt thereof.

[0009] To achieve another objective of the present invention, the present invention provides a health functional food composition for the prevention or improvement of heart disease comprising bindarite or a food-grade acceptable salt thereof.

[0010] To achieve another objective of the present invention, the present invention provides a composition for protecting endothelial cells (EC) comprising vindarit.

[0011]

[0012] The present invention will be described in detail below.

[0013] In one aspect of the present invention, the invention relates to a pharmaceutical composition for the prevention or treatment of heart disease comprising vindarite or a pharmaceutically acceptable salt thereof.

[0014] The above-mentioned Bindarit ((2-((1-Benzyl-1H-indazol-3-yl)methoxy)-2-methylpropanoic acid)) is an NSAID targeting CCL2, known as a treatment for nephropathy, autoimmune diseases, and ocular diseases, and is a substance represented by the following chemical formula.

[0015]

[0016] In the present invention, the "heart disease" is a heart disease that can be caused by various factors such as damage to endothelial cells (EC), inflammation, and death of myocardial cells. More specifically, it may include myocardial infarction (MI), ischemic cardiomyopathy, cardiac hypertrophy, myocardial contractility disorder, myocardial ischemia, heart failure, and angina pectoris, and preferably may be myocardial infarction or ischemic cardiomyopathy.

[0017] In one embodiment of the present invention, it was confirmed that the vindarit of the present invention has a significant effect on the protection of endothelial cells (ECs) against cellular stress and apoptosis induced particularly by tumor necrosis factor alpha (TNF-α), that is, on the proliferation and growth of said ECs. Furthermore, through this effect, it may have the effect of preventing and treating heart diseases caused by factors such as myocardial infarction (MI), cardiac fibrosis, and myocardial cell damage, or alleviating cardiac dysfunction resulting therefrom.

[0018] In particular, patients with myocardial infarction (MI) have severe damage to vascular endothelial cells, which can cause severe inflammation upon reperfusion. Vindarit of the present invention can reduce the size of myocardial infarction and suppress damage upon reperfusion by promoting the proliferation and growth of endothelial cells (ECs) and inhibiting apoptosis through an endothelial cell protective effect. Additionally, vindarit can have the effect of treating ischemic heart diseases such as angina pectoris or suppressing ischemic tissue damage by restoring endothelial function and protecting endothelial cells.

[0019] In this specification, the term "prevention" refers to any act that can suppress heart disease and cardiac dysfunction or delay its onset by administering the pharmaceutical composition according to the present invention.

[0020] In this specification, the term "treatment" refers to any act in which symptoms associated with heart disease and cardiac dysfunction are improved or benefited by the administration of the pharmaceutical composition according to the present invention.

[0021] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods, and may additionally include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may additionally be included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0022] For example, solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, or gelatin, with the extract or compound. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0023] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0024] The pharmaceutical composition of the present invention may be administered orally or parenterally (intravenous injection, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time, and may be selected in an appropriate form by those skilled in the art.

[0025] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" refers to a reasonable amount applicable to medical treatment, meaning an amount sufficient to treat a disease, and the criteria may be determined based on the patient's disease, severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant components, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage may be determined to a level that minimizes side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and generally, an amount of 0.001 to 150 mg, more preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, 1 to 3 times a day. However, this is for illustrative purposes only, and the above dosage may be set differently as needed.

[0026] Furthermore, the present invention relates to a food or health functional food for the prevention or improvement of heart disease comprising bindarit or a food science-acceptable salt thereof. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body pursuant to Article 6727 of the Health Functional Foods Act, and the term "functional properties" means consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0027] The food or health functional food of the present invention can be manufactured and processed into pharmaceutical administration forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or into health functional foods such as tea bags, infusions, beverages, candies, jellies, and gums for the purpose of preventing and improving heart disease.

[0028] The food or health functional food composition of the present invention may be used as a food additive and may be manufactured into a product either alone or in combination with other ingredients. Additionally, it may include nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents and promoters, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The above ingredients may be used alone or in combination, and may be combined and used in appropriate amounts.

[0029] In another aspect of the present invention, the present invention relates to a composition for protecting endothelial cells (EC) comprising vindarit.

[0030] The term "endothelial cell protection" refers to enhancing the survival and proliferation of endothelial cells and protecting them from apoptosis. These endothelial cells (ECs) play a pivotal role in the recovery of cardiac function after myocardial infarction (MI) through vascular recanalization and improved perfusion of the infarct site. Furthermore, endoplasmic reticulum (ER) stress signals play a crucial role in regulating cell survival and death, and the vindarit of the present invention has been identified to play a role in regulating these ER stress signals.

[0031] The endothelial cell protective composition of the present invention may be prepared in the form of a food composition or a food additive, and in particular, may be prepared in the form of a health food composition. The food composition is as described above.

[0032] The endothelial cell protective composition of the present invention can increase the survival and proliferation of endothelial cells, further improve vascular recanalization and infarction reperfusion after cardiac infarction (MI), and influence neovascularization in the affected area to help restore cardiac function.

[0033]

[0034] The present invention relates to a composition for the prevention, improvement, or treatment of heart disease comprising vindarit, wherein the composition improves the survival and proliferation of endothelial cells (ECs) of the heart and further protects them from apoptosis, thereby having preventive, improvement, and therapeutic effects for heart disease.

[0035] Figure 1 shows the results of qRT-PCR analysis on Prmt7, Ki67, and Vegfα mRNA expression levels after treating C166 cells with various concentrations of vindarit for 24 hours.

[0036] Figure 2 shows the experimental process for observing cardiac function in a group treated with vindarit for 21 days (3 weeks) in mice that had MI (myocardial infarction) induced in mice.

[0037] Figure 3 shows the results of measuring the improvement in cardiac function after 1 week and 3 weeks by the Bindarite treatment in the experiment of Figure 2 above.

[0038] Figure 4 shows representative images of cardiac ultrasound examinations of mice at 1 week (A) and 3 weeks (B) after MI, and the results of measuring the relative cardiac mass to body weight and electrocardiogram parameters (EF, FS) at 1 week (A) and 3 weeks (B) after MI.

[0039] Figures 5A and 5B show hematoxylin and eosin (H&E) and Mason trichrome (MT) stained images (scale bar, 50 μm) of heart sections of mice 3 weeks after MI, and the quantification of the percentage of necrotic and fibrotic areas of the heart sections.

[0040] Figure 6 shows the results of immunoblotting analysis on Collagen I (Col I), Collagen III (Col III), cardiac troponin I (cTnI), and Gapdh protein levels in cardiac lysates of mice induced with MI after 3 weeks.

[0041] Figure 7 is an enrichment map comparing gene ontology (GO) terms between a vehicle and a Vindarit-administered MI heart, where nodes represent sets of genes connected by lines and grouped into sub-clusters.

[0042] Figure 8 shows representative gene ontology (GO) terms of subclusters in response to stress, and the gene interaction network was constructed from a set of "response to stress" genes with lines indicating interaction types.

[0043] Figure 9 shows the results of performing Cytoscape for ontology clustering. GO terms are represented as nodes, and the node size indicates the significance of term enrichment. Groups with fewer than two connections were excluded from the final network list, representative genes of sub-clusters are displayed on the plot, and lines and shapes represent the types of interaction and regulation.

[0044] Figure 10 shows representative immunofluorescence images (scale bar, 20 μm) of Tnnt2 (red), cleaved caspase 3 (c-cas3, green), and DAPI in heart sections of mice 3 weeks after Sham and MI induction, and quantification of the mean intensity of cleaved caspase 3 (c-cas3) expression in heart sections.

[0045] Figure 11 shows the results of immunoblotting analysis for Atf4, Chop, p-eIF2α, eIF2α, p53, Prmt7, and Gapdh protein levels in cardiac lysates of Sham, MI, and EndoKO-MI mice.

[0046] Figure 12 shows the results of quantifying Atf4, Chop, p-eIF2α, p53, and Prmt7 protein levels in cardiac lysates of Sham, MI, and MI-vindarit mice.

[0047] Figure 13 shows representative immunofluorescence images showing the expression of Chop (red), Cdh5 (green), and DAPI (blue) in heart sections of Sham, MI, and EndoKO-MI mice, and quantification of the average intensity of Chop.

[0048] Figure 14 shows representative confocal images (scale bar, 10 μm) showing Chop (green) expression and DAPI (blue) staining in C166 cells treated for 24 hours with DMSO, TNFα (50 ng / ml), Bindarit (500 nM), or a combination of TNFα and Bindarit, and the results of quantifying the average intensity of Chop expression in C166 cells.

[0049] Figure 15 shows the results of an immunoblotting analysis of p-eIF2α, eIF2α, Atf4, Chop, and Gapdh protein levels in C166 cells treated for 24 hours with DMSO, TNFα (50 ng / ml), vindarit (500 nM), or a combination of TNFα and vindarit.

[0050] Figure 16 shows the results of qRT-PCR analysis on Ki67, Icam1, Vcam1, Il1α, and Il6 mRNA expression levels in C166 cells treated for 3 hours with DMSO, TNFα (50 ng / ml), vindarit (500 nM), or a combination of TNFα and vindarit.

[0051] Figure 17 shows representative images of BrdU staining (red) and DAPI (blue) in C166 cells treated for 24 hours with DMSO, TNFα (50 ng / ml), vindarite (500 nM), or a combination of TNFα and vindarite, and the results of quantifying the percentage of BrdU-positive cells in C166 cells.

[0052] Figure 18 shows representative images of scratch wound healing assays in C166 cells treated for a specified time with DMSO, TNFα (50 ng / ml), vindarite (500 nM), or a combination of TNFα and vindarite, and results of quantifying the relative wound closure area in C166 cells based on the scratch wound healing assay results.

[0053] Figure 19 shows microscopic images of the tube formation assay in C166 cells treated for 24 hours with DMSO, TNFα (50 ng / ml), vindarite (500 nM), or a combination of TNFα and vindarite, and quantifies the number of tubes formed as a result of the tube formation assay in C166 cells.

[0054] Figure 20 shows representative immunofluorescence images (scale bar, 20 μm) of Cdh5 (green), Tnnt2 (red), and DAPI (blue) in heart sections and the results of quantifying the number of capillaries in heart sections.

[0055] Hereinafter, embodiments are described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those with average knowledge in the art.

[0056]

[0057] Experimental Methods and Materials

[0058] 1. Preparation of animal models for mouse experiments

[0059] To evaluate the role of endothelial Prmt7 in CVD, this study used approximately 3-month-old male cadherinous mice born from heterozygous mating. To induce Prmt7 ablation in EC, Prmt7fl / fl;VE-cadherin-ERT2Cre mice were treated with tamoxifen (Tmx) for 2 weeks.

[0060] The MI mouse model was established via left coronary artery (LCA) ligation without mechanical ventilation, as previously reported, and LCA ligation was performed on 10-week-old mice. The control group underwent the same surgical procedure without closing the LCA. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University College of Medicine and was conducted in accordance with ethical guidelines (Protocol No.: SKKUIACUC2023-02-21-1).

[0061] 2. Echocardiogram Analysis

[0062] Mice were anesthetized with 1% (vol / vol) isoflurane, and heart rates were measured 1 day, 1 week, and 3 weeks after MI using a Vevo LAZR-X photoacoustic imaging system (Fujifilm Visual Sonics). Heart rate was monitored and generally maintained at 400–500 beats per minute. Ejection fraction (EF) and fractional shortness (FS) were calculated by performing M-mode image analysis derived from a short-shortened view of the left ventricle (LV).

[0063] 3. Histology, Immunohistochemistry, and Immunofluorescence

[0064] Histology of heart sections was performed according to known methods. Harvested mouse hearts were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin blocks or Tissue-Tek Optimal Cutting Temperature (OCT) compounds (Sakura Finetec). Paraffin-embedded or frozen heart tissues were sectioned to a thickness of 7 μm, and histological analyses such as hematoxylin and eosin (H&E, BBC biochemical) and Masson's tricolor (Abcam) were performed. Images were captured using a Nikon ECLIPS TE-2000U inverted microscope and Tissue FAXS imaging software (TissueGnostics).

[0065] Immunohistochemical staining of cardiac samples was performed using known methods. Fluorescence images were obtained using an LSM-710 confocal microscope system (Carl Zeiss) or a Cytation C10 confocal microscope system (Agilent). Images were analyzed using Image J software to quantify signal intensity. For immunofluorescence staining, cardiac sections were fixed with 4% paraformaldehyde (PFA) and then permeated with 0.5% Triton X-100 to facilitate antibody penetration. The antigen was recovered by immersing the sections in a sodium citrate solution at pH 6.0 and heating them while boiling for 10 minutes, followed by recovery with a 5% goat serum solution. Subsequently, the immunostains were cultured and visualized using the antibodies listed in Table 1 below.

[0066] AntigenCat No.ManufacturerGAPDHLF-PA0018AbFrontierPrmt7Sc98882Santa Cruzp-eIF2α3398SCell SignalingIF2α5324SCell Signalingp53sc-126Santa Cruzc-cas 39664SCell SignalingCollagen IAB765PChemiconCollagen IIIab184993AbcamcTnIMA1-20112InvitrogenBrdUsc-32323Santa Cruz

[0067] 4. Evans Blue Hair Dye

[0068] Evans Blue staining was used to evaluate cell damage in the myocardium according to the manufacturer's instructions. Evans Blue (Sigma-Aldrich) was dissolved in PBS (10 mg / ml) and sterilized by passing it through a membrane filter with a pore size of 0.2 μm. Then, the Evans Blue solution was injected into mice via the tail vein at a concentration of 1% w / v, and 3 hours after injection, the mice were sacrificed and the uptake of the dye into the myocardium was visually inspected and indicated.

[0069] 5. TUNEL Assay

[0070] TUNEL imaging analysis (Click-iT plus TUNEL Assay) was performed to investigate myocardial cell apoptosis. Briefly, cardiac sections were permeated, and a mixture of terminal deoxynucleotide transferase reaction buffer and EdUT was applied to the sections. After 60 minutes of incubation, the sections were rinsed in PBS with 3% BSA and 0.1% Triton X-100 for 5 minutes, treated with a TUNEL reaction cocktail mixture for 30 minutes, and mounted with a mounting solution containing DAPI (Abcam) to visualize the nuclei. Images were analyzed using a Cytation C10 confocal microscope system (Agilent).

[0071] 6. Cell Culture

[0072] C166 cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin under standard culture conditions (37°C and 5% CO2). To evaluate the inhibitory effect on Prmt7 under stress, cells were treated with 1 μM SGC8158 (Sigma-Aldrich), a Prmt7 inhibitor, along with 0.2% BSA or 50 ng / ml TNFα (PeproTech); to promote Prmt7 overexpression, cells were treated with Vindarit (Sigma-Aldrich, 500 nM). Cell viability was assessed using the 3-(4,5-Di-2-yl)-2,5-ditetrazolium bromide (MTT) assay. C166 cells were cultured in 96-well plates at a density of 5 x 10⁴ per well. 3 After dispensing at cell density and incubating for 24 hours, the cells were incubated with 0.5 mg / mL MTT solution (Sigma-Aldrich) for 4 hours. Then, the medium was removed, 100 μL of DMSO was added to each well, and incubated for 10 minutes. Absorbance was measured at 570 nm using a spectrophotometer (Thermo Scientific).

[0073] 7. Bromodeoxyuridine (BrdU) staining

[0074] To investigate cell proliferation characteristics, C166 cells were grown on chamber slides at appropriate confluence points. After treatment with various conditions, cells were cultured in fresh medium containing BrdU at 10 μM for 4 hours, fixed in cold methanol (-20°C) for 4 minutes, and then denatured in 2 M hydrochloride at room temperature for at least 20 minutes. Cells were blocked with 3% BSA in PBS at room temperature for 1 hour, treated with the primary anti-BrdU antibody and the Alexa Fluor 546 secondary antibody, and then mounted using a mounting solution containing DAPI (Abcam). Images were analyzed using a Cytation C10 confocal microscope system (Agilent).

[0075] 8. Scratch Analysis

[0076] 5x10 to test mobility 4 C166 cells were seeded into 6-well plates and grown until they reached approximately 90% confluence. Mechanical scratches were made in the cell monolayer using a pipette tip. Images were then captured after incubation for various durations (0, 12, 24, 36, and 48 hours), and migration characteristics were quantified using the percentage of wound closure.

[0077] 9. Analysis of tubularity

[0078] 5X10 to evaluate the angiogenic potential of endothelial cells in vitro 4 C166 cells were inoculated into a Matrigel-coated 24-well plate and formed capillary-like structures for 2 hours.

[0079] 10. Protein Analysis

[0080] Immunoblotting analysis was performed in a known manner. Briefly, lysed cardiac tissue or cultured cells were lysed using a lysis buffer composed of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and a complete protease inhibitor cocktail (Roche Diagnostics). Protein concentrations were measured at 562 nm using a spectrophotometer with a BCA protein assay reagent (Pierce). Cell lysates were applied to SDS-PAGE (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with Tris-buffered saline (TBST) and 5% bovine serum albumin for 30 minutes and incubated overnight at 4°C with the primary antibody. The blots were washed with TBST for 20 minutes, incubated with a peroxidase-conjugated secondary antibody at room temperature for 1.5 hours, washed with TBST for 20 minutes, and visualized. Protein-level quantification was obtained by signal intensity analysis using the Image J (NIH) program and normalized to a loading control.

[0081] 11. RNA Analysis

[0082] qRT-PCR and RNA sequencing analysis were performed according to known methods. Total RNA from mouse heart and C166 cells was extracted using easy-BLUE (iNtRON) reagents according to the manufacturer's instructions. Primer sequences for qRT-PCR are listed in Table 1 below. cDNA samples were generated from 0.5 μg of RNA using the PrimeScript RT reagent kit (TaKaRa) according to the manufacturer's protocol. RNA sequencing analysis was performed using an RNA 6000 Nano Chip (Agilent Technologies) with an Agilent 2100 bioanalyzer.

[0083] Analysis of RNA sequencing data was performed using ExDEGA v1.61 (e-Biogen) and the ClueGO / CluePedia, EnrichmentMap, and GeneMania plugins of Cytoscape (v3.10.0) software. Total gene expression was evaluated as a biological process using Gene Set Enrichment Analysis with the MSigDB database v6.1 (>1.3-fold, RC log2>2, P<0.05).

[0084] Gene symbol5' to 3'Prmt7Forward5'-TTC-CCA-CAG-CGG-GCA-TTA-T-3'Reverse5'-TGT-AGC-ATG-TCG-GCA-TAG-GA-3'Ki67Forward5'-TCA-TGA-GGA-TGG-AAG-CAA-GCC-3'R everse5'-CTC-ACT-CTT-CTC-AGG-GTC-AGC-A-3'VcamForward5'-GCC-ACC-CTC-ACC-TTA-ATT-GCT-ATG-3'Reverse5'-TGT-GCA-GCC-ACC-TGA-GAT-CC-3'G apdhForward5'-GAC-ATG-CCG-CCT-GGA-GAA-AC-3'Reverse5'-AGC-CCA-GGA-TGC-CCT-TTA-GT-3'IL-1αForward5'-GGA-GAA-GAC-CAG-CCC-GTG-TTG-CT-3 'Reverse5'-CCG-TGC-CAG-GTG-CAC-CCG-ACT-T-3'IL6Forward5'-CAA-CGA-TGA-TGC-ACT-TGC-AGA-3'Reverse5'-GTG-ACT-CCA-GCT-TAT-CTC-TTG-GT-3'

[0085] 12. scRNAseq data analysis

[0086] scRNA-seq data previously deposited with GEO under accession number GSE201947 and snRNA-seq data published on the Zenodo Data Archive (https: / / zenodo.org / record / 6578047) were used to further evaluate the role of Prmt7 in EC cells. All scRNA-seq datasets were SeqGeq TM Analysis was performed using a program. The datasets were integrated using the dimensionality reduction platform's pipeline. The integrated cells underwent a quality control process to remove dead cells and doublets based on cells representing library size and variance. The integrated cells were then normalized to counts per million (CPM). The filtered cells were then clustered using the Seurat pipeline with a resolution of 0.5. To visualize the data, dimensionality reduction UMAPs were generated via the Seurat function RunUMAP. Cluster identities were assigned based on the expression of top marker genes and marker genes known from the literature. Genes with differential expression in the scRNAseq database were extracted based on the conditions of 1) p-value <0.05 and 2) fold change > 1.5. For PCA analysis, selected genes from each group in the RNAseq samples were extracted based on p-value <0.05 and log2 expression > 2.0, and were automatically clustered into 20 variances using SeqGeq software.

[0087] 13. Gene Ontology (GO) Enrichment Analysis of DEG

[0088] Enrichment pathways associated with the biological pathway database were visualized using the Cytoscape (v3.1.0) cluego plugin. Briefly, biological GO terms were explored with moderate specificity and a kappa score of 0.4. The enrichment / depletion method using a two-sided hypergeometric test was applied Bonferroni stepwise for each p-value calculation, and enrichment pathways with a p-value less than 0.05 were considered significant. Gene Set Enrichment Analysis (GSEA) was performed to extract knowledge regarding overexpressed gene ontology terms for various functional processes and signaling pathways across samples. Visualization of significantly enriched GO terms for functional processes and signaling pathways across samples was performed using the Cytoscape plugin EnrichmentMap. Mapping of gene expression levels was performed using the GeneMania plugin. In the analysis, all GO terms in the network were filtered based on pathway scores with a p-value less than 0.05.

[0089] 14. Statistical Analysis

[0090] All experiments were repeated at least three times with identical or similar results. Data represent biological replicates. Appropriate statistical analysis was performed for each analysis. The data satisfied the hypotheses of the statistical tests described for each experiment. The statistical significance of the differences was determined according to GraphPad Prism 8.4.3 (GraphPad Software Inc., San Diego, California, USA), and P-values ​​*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant.

[0091]

[0092] Example 1. Regulation of expression of stress markers, mitochondrial activity, and genes related to vascular endothelial cell proliferation by administration of Vindarit

[0093] 1-1. Effect of Vindarite Treatment on Cardiac Function in MI Models

[0094] Experiments were conducted to confirm the effect of vindarit treatment on improving cardiac function in a myocardial infarction model. First, it was confirmed that the expression of Prmt7 in endothelial cells increased upon vindarit treatment, and that the maximum Prmt7 induction was at a concentration of 1 μM (Fig. 1A). In addition, upon vindarit treatment, the expression of proliferation regulators Ki67 and Vegfa was increased, indicating that they play a potential role in angiogenesis.

[0095] Based on this, the effects of vindarit on cardiac dysfunction in a myocardial infarction model were confirmed, and the experimental procedure is shown in Fig. 2. According to the experimental method described above, electrocardiograms were performed on mice at weeks 1 and 3 after MI induction, and the results are shown in Fig. 3. In Fig. 3, MI mice treated with vindarit showed milder changes in HR compared to MI mice treated with a vehicle. Additionally, while mice treated with a vehicle showed significant changes in cardiac rhythms (RR, SDNN, QRS, and QT), it was confirmed that the incidence of arrhythmia decreased at week 3 after MI when vindarit was administered. Furthermore, the echocardiogram results in Fig. 4A showed that at week 1 after MI, both MI groups experienced a significant decrease in EF and FS compared to the Sham group, but the degree of decrease was much milder in mice treated with vindarit compared to mice treated with a vehicle. Furthermore, regarding the cardiac mass of MI-induced mice, it was confirmed that while the relative cardiac mass of the control MI group was significantly reduced, this change was alleviated upon administration of vindarit. Additionally, as shown in Figure 4B, 3 weeks after MI, the MI group treated with vindarit showed improved cardiac function approaching the level of the sham group, whereas the control MI group experienced a severe decrease in EF and FS. Moreover, the relative cardiac mass of the control MI group increased significantly compared to the sham group, but this was alleviated by the administration of vindarit (Figure 4B).

[0096] Histological staining confirmed that treatment with vindarit prevented hypotrophy of myocardial cells, which was manifested by a reduction in the cross-sectional area of ​​myocardial cells and the area of ​​necrosis and fibrosis induced by MI (Figs. 5A and 5B). Similarly, while marker proteins for fibrosis (collagen I and III) and myocardial cell damage (cardiac troponin I) were significantly increased in MI hearts, these levels were alleviated upon treatment with vindarit (Fig. 6).

[0097] 1-2. Changes in Gene Expression Caused by Vindarite Treatment

[0098] RNA sequencing was performed to investigate the fundamental molecular changes caused by vindarit treatment. Detailed analysis of the dataset of Vehicle and vindarit-treated MI hearts revealed that genes related to stress, cycle g2 transition, actin cytoskeletal tissue, growth and development, wound healing, cardiac blood circulation, striated muscle contraction, mitochondrial translation, activated Mapk cascade, and immune response were upregulated (Fig. 7).

[0099] Analysis of gene subclusters in response to stress revealed that genes related to the response to lipids, cellular response to nitrogen compounds, regulation of the transmembrane receptor protein serine threonine kinase signaling pathway, cellular response to reactive oxygen species, response to calcium ions, response to nutrients, regulation of cellular response to insulin stimulation, response to BMPs, response to endogenous stimuli, and regulation of oxidative stress-induced apoptosis were altered (Fig. 8).

[0100] Cytoscap for ontology clustering confirmed that genes associated with the negative regulation of vascular endothelial cell proliferation, vascular smooth muscle cell proliferation, energy homeostasis, response to muscle elongation, ATPase-binding cation transmembrane transporter activity, and focal adhesion assembly in vindarit-MI hearts belong to an upregulated ontology (Fig. 9). In particular, treatment with vindarit upregulated several important genes involved in cardiac repair, such as Mef2c, Ncam1, Igf1, and Pdgfα, through the regulation of revascularization, apoptosis, fibrosis, and EC homeostasis.

[0101] In conclusion, the cardioprotection mediated by vindarit treatment is likely due to the induction of genes important for inhibiting apoptosis and promoting EC proliferation, contributing to the revascularization of the infarct site.

[0102]

[0103] Example 2. ER stress relief effect

[0104] 2-1. Changes in ER Stress-Related Protein Expression Induced by Vindarit Administration in an MI Model

[0105] Consistent with the transcriptome data above, administration of vindarit to MI hearts reduced c-cas3 immunopositivity (Fig. 10), and the increase in p-eIFα, Chop, and p53 proteins was attenuated in the vindarit-treated group compared to MI hearts treated with a vehicle (Figs. 11 and 12). Additionally, Chop expression in Cdh5-positive endothelial cells in MI hearts was significantly increased, which was attenuated in MI hearts treated with vindarit (Fig. 13).

[0106] 2-2. Effect of Vindarit Administration on ER Stress Relief in In Vitro

[0107] To confirm the protective effect of vindarit on endothelial cells, C166 cells were induced with ER stress by treating them with TNFα and then treated with vindarit. TNFα treatment increased Chop protein expression in endothelial cells, but this increase was attenuated upon co-treatment with vindarit (Fig. 14). Similarly, TNFα treatment increased ATF4, p-eIF2α, and Chop proteins in endothelial cells; however, following vindarit treatment, p-eIF2α and Chop expression increased, while ATF4 protein remained unchanged (Fig. 15).

[0108] The above results suggest that the administration of vindarit plays an important role in regulating ER stress-induced apoptosis in cardiac and endothelial cells caused by MI.

[0109]

[0110] Example 3. Endothelial cell (EC) protective effect

[0111] 3-1. Confirmation of Endothelial Cell (EC) Survival and Proliferation Marker Expression

[0112] To confirm the protective effect of vindarit on endothelial cells, endothelial cells were pretreated with vehicle or vindarit at a concentration of 0.5 μM for a total of 24 hours.

[0113] TNFα was administered at a concentration of 50 ng / ml during the last 3 hours of vehicle or vindarit administration. Upon administration of vindarit, the inhibition of Ki67 expression induced by TNFα treatment was alleviated, while the increase in endothelial cell dysfunction and inflammatory markers Icam1, Vcam1, Il-1α, and Il-6 was alleviated (Fig. 16). Similarly, upon administration of vindarit, the decrease in EC proliferation induced by TNFα administration was alleviated (Fig. 17). Furthermore, upon administration of vindarit, the effect of stimulating EC migration was confirmed under both normal and TNFα-induced stress conditions (Fig. 18). Taken together, these data suggest that vindarit plays an important role in the survival and proliferation of ECs and may contribute to revascularization after ischemic injury by protecting ECs from TNFα-induced apoptosis.

[0114] 3-2. Effects of Vascular Damage Reduction and Revascularization Promotion in an MI Model

[0115] In addition, regarding the damage to endothelial cell tubule formation caused by TNFα administration, it was confirmed that this damage was alleviated when vindarit was administered (Fig. 19).

[0116] To evaluate revascularization in an MI model, the capillary density of the heart was examined 3 weeks after MI induction. 3 weeks after MI, the heart showed a significant increase in Cdh5-positive endothelial cells compared to the sham heart (Fig. 20). This suggests that revascularization is important for myocardial infarction recovery. When vindarit was administered, Cdh5-positive endothelial cells increased further, confirming that vindarit administration can contribute to improving recovery after MI injury by further increasing the vascular density of the MI heart.

[0117] In summary, Prmt7 deficiency in endothelial cells can cause cardiac damage exacerbated by ischemia resulting from unregulated ER stress responses and apoptosis, and vindarit can improve cardiac cell viability by inducing the expression of Prmt7 and improve post-ischemic cardiac recovery through revascularization.

[0118]

[0119] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. A pharmaceutical composition for the prevention or treatment of heart disease comprising Bindarit or a pharmaceutically acceptable salt thereof.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of heart disease, wherein the heart disease is selected from the group consisting of myocardial infarction (MI), ischemic cardiomyopathy, cardiac hypertrophy, myocardial contractility disorder, myocardial ischemia, heart failure, and angina pectoris.

3. In Paragraph 1, The above composition is a pharmaceutical composition having the effect of promoting the survival and proliferation of endothelial cells (EC).

4. In Paragraph 1, The above composition is a pharmaceutical composition having an endoplasmic reticulum (ER) stress-relieving effect.

5. In Paragraph 1, The above composition is a pharmaceutical composition having an effect of preventing blood vessel damage or regenerating blood vessels.

6. A health functional food composition for the prevention or improvement of heart disease comprising Bindarit or a food-grade acceptable salt thereof.

7. In Paragraph 6, A health functional food composition for the prevention or improvement of heart disease, wherein the heart disease is selected from the group consisting of myocardial infarction (MI), ischemic cardiomyopathy, cardiac hypertrophy, myocardial contractility disorder, myocardial ischemia, heart failure, and angina pectoris.

8. In Paragraph 6, The above composition is a health functional food composition having the effect of promoting the survival and proliferation of endothelial cells (EC).

9. In Paragraph 6, A health functional food composition having an endoplasmic reticulum (ER) stress-relieving effect.

10. In Paragraph 6, The above composition is a health functional food composition having the effect of preventing blood vessel damage or regenerating blood vessels.

11. Bindarit or a pharmaceutically acceptable salt thereof used for the prevention or treatment of heart disease.

12. In Paragraph 11, Bindarit or a pharmaceutically acceptable salt thereof, characterized in that the heart disease is selected from the group consisting of myocardial infarction (MI), ischemic cardiomyopathy, cardiac hypertrophy, myocardial contractility disorder, myocardial ischemia, heart failure, and angina pectoris.

13. A method for treating heart disease comprising the step of administering an effective amount of Bindarit or a pharmaceutically acceptable salt thereof to a subject.

14. In Paragraph 13, A treatment method characterized in that the heart disease is selected from the group consisting of myocardial infarction (MI), ischemic cardiomyopathy, cardiac hypertrophy, myocardial contractility disorder, myocardial ischemia, heart failure, and angina pectoris.

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