Application of AMBP in prevention and treatment of aortic valve calcification by inhibiting FHL3-mediated ERK1 / 2 and JNK pathways

Abstract

The invention discloses an application of AMBP in prevention and treatment of aortic valve calcification by inhibiting FHL3 mediated ERK1 / 2 and JNK pathways, the AMBP is a promising therapeutic target, and the progress of CAVD is relieved by protecting valvular interstitial cells from pathological calcium deposition and osteogenic transformation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of AMBP in preventing and treating aortic valve calcification by inhibiting ERK1 / 2 and JNK pathways mediated by FHL3. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Calcific aortic valve disease (CAVD) is a progressive disease characterized by aortic valve (AV) calcification and fibrosis. Despite advances in our understanding of the pathogenesis of CAVD, no drug has been shown to be effective in preventing AV calcification.

[0004] As the aging population continues to grow worldwide, the prevalence of CAVD has increased significantly, making it the most common acquired valvular heart disease. Calcific aortic stenosis (AVS) is a late manifestation of CAVD, characterized by progressive fibrocalcification and thickening of the aortic valve leaflets, ultimately leading to valve obstruction.

[0005] CAVD is considered an active, multifactorial pathogenic process that is caused by pathological changes in aortic valve cells, leading to matrix remodeling, subsequent valve calcification, and hemodynamic obstruction. This process is generally divided into two stages: an initial stage with lipid deposition, endothelial injury, dysfunction, and inflammation, and a progressive stage with the differentiation of valve interstitial cells into myofibroblasts and osteoblast-like cells, activating calcification pathways. These events ultimately lead to end-stage calcification of the valve.

[0006] With the innovative development of cardiovascular interventional technology, transcatheter active valve replacement, as a representative interventional aortic valve treatment method, has been widely promoted and applied worldwide. However, this treatment method can only be used as a supplementary treatment measure for CAVD, and the service life of the artificial valve is limited. Risk factors for CAVD, such as smoking, hypertension, elevated low-density lipoprotein cholesterol (LDL-C), high lipoprotein (a) levels, and aging, are similar to those associated with atherosclerosis. However, in clinical practice, coronary artery disease and CAVD do not always occur at the same time, and drug treatments for these conditions do not produce similar results. Multiple randomized clinical trials have shown that there is no significant difference between statins and placebo in slowing the progression of aortic valve calcification, indicating that statins are ineffective in the progressive stage of CAVD. This ineffectiveness may be due to the difficulty in reversing membrane calcification at this late stage, which implies the existence of a lipid-dependent mechanism. Therefore, elucidating the mechanism of CAVD is crucial for developing intervention strategies aimed at preventing or slowing the progression of CAVD. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide an application of AMBP in preventing and treating aortic valve calcification by inhibiting the ERK1 / 2 and JNK pathways mediated by FHL3.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides use of a substance that promotes overexpression of AMBP in the preparation of aortic valve calcification and / or fibrosis products.

[0010] In a second aspect, the present invention provides the use of a substance that promotes overexpression of AMBP in the preparation of a product that inhibits the phosphorylation of ERK1 / 2 and JNK in VIC cells.

[0011] In a third aspect, the present invention provides a pharmaceutical composition comprising the substance that promotes overexpression of AMBP and / or at least one other non-drug active ingredient.

[0012] In some embodiments, the non-pharmaceutical active ingredient is a carrier, excipient or diluent commonly used in pharmacy.

[0013] Preferably, the carrier, excipient or diluent is selected from at least one of calcium silicate, lactose, sucrose, sorbitol, erythritol, cellulose, calcium phosphate, mannitol, starch, gum arabic, methylcellulose, gelatin, glucose, maltitol, talc, xylitol, polyvinyl pyrrolidone, microcrystalline cellulose, methyl hydroxybenzoate, alginate, propyl hydroxybenzoate, water, magnesium stearate or mineral oil.

[0014] In some embodiments, the pharmaceutical composition is in the form of an external preparation, an oral preparation, a suppository or an injection.

[0015] Preferably, the oral dosage form is a tablet, powder, syrup, suspension, granule, emulsion, capsule or spray.

[0016] In a fourth aspect, the present invention provides use of the pharmaceutical composition in preparing aortic valve calcification and / or fibrosis products.

[0017] In some embodiments, the product is a drug or a test reagent.

[0018] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0019] Calcific aortic valve disease (CAVD) is characterized by progressive aortic valve calcification and fibrosis, but there is currently no effective drug treatment. This study used ApoE induced by a high cholesterol diet to treat CAVD. - / -A CAVD model was established in mice, and AMBP was identified as a key regulatory factor in CAVD, and its mechanism of action was explored. AMBP overexpression significantly reduced aortic valve calcification and fibrosis, and reversed osteogenic differentiation and calcium deposition of valve interstitial cells.

[0020] Mechanistically, AMBP competitively binds to the zinc finger domain of FHL3, disrupting the protective effect of FHL3 on the stability of phosphorylated ERK1 / 2 (P-ERK1 / 2) and JNK (P-JNK). This competitive inhibition prevents osteoblast differentiation by inhibiting the P-ERK1 / 2 and P-JNK signaling pathways. Bioinformatics analysis, AlphaFold3-based simulations, and co-immunoprecipitation confirmed this interaction. These findings suggest that AMBP is a promising therapeutic target to mitigate the progression of CAVD by protecting valvular interstitial cells from pathological calcium deposition and osteogenic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 .CAVD patients and ApoE - / -AMBP expression was increased in calcified AVs of mice. (A) Volcano plot. Blue, red, and gray dots represent genes with downregulated expression, upregulated expression, and genes with no significant change in expression in the CAVD and non-CAVD groups. The screening parameters were |log2(FC)|>1 and q value (FDR-adjusted p value) <0.05. (B) Heat map of the top 50 DEGs; red and blue represent high expression and low expression, respectively. (C) Venn diagram validates the overlap of hub genes in the five algorithms. (D) qRT-PCR detection of mRNA expression of key hub genes in aortic valve tissues of CAVD (n=5) and non-CAVD (n=4) groups: (a) AMBP, (b) FGG, (c) FGA, (D) SERPINC1, (e) APOA2, (f) APOB, and (g) ACAN. (E-F) Representative immunofluorescence images (E) and quantitative analysis (F) of VICs (green) in aortic valve tissues of CAVD (n = 5) and non-CAVD (n = 4) groups (scale bar = 50 μm). (G) Time course of CAVD modeling in ApoE- / - mice. (H) Representative immunohistochemical images (scale bar = 0.2 mm) and quantitative analysis of AMBP expression in AV tissues of HCD and ND mice (n = 6). (I) Representative immunoblot images and quantitative analysis of the temporal expression pattern of AMBP in VICs under OM induction (n = 6). Values ​​are expressed as interquartile range (D) or mean ± SEM (F, H, and I). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Abbreviations: AMBP, alpha-1 microglobulin / bikunin precursor; AAV, aortic valve; CAVD, calcific aortic valve disease; DEGs, differentially expressed genes; FC, fold change; FDR, false discovery rate; HCD, high cholesterol diet; ND, normal diet; OM, osteogenic medium; qRT-PCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; VICs, valvular interstitial cells.

[0023] Figure 2 AMBP overexpression alleviates HCD-induced ApoE - / - Aortic valve calcification in mice. (A) HCD-induced ApoE - / - Schematic diagram of the time course of mouse aortic valve calcification. (BC) qRT-PCR detection of ApoE - / - Expression of Ambp (B), Runx2 and Osterix (C) mRNA in mouse aortic valve tissue (n=6). (D) Representative HE images and ApoE - / -Quantitative analysis of aortic valve thickening in mice (n=4, scale bar=0.05 mm). (E) Representative Masson staining images and quantitative analysis (n=4, scale bar=0.2 mm). (F) Representative Alizarin Red S (black arrows indicate calcified lesions) and Von Kossa (red arrows indicate calcified lesions) staining images and quantitative analysis of aortic valve calcification (n=4, scale bar=200 μm). (G) Representative images of aortic valve pulsed Doppler blood flow spectra and quantitative analysis of transvalvular peak jet velocity and aortic valve peak pressure in echocardiography (n=6). Values ​​are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Abbreviations: AMBP, alpha-1-microglobulin / bikunin precursor; AAV-Nc, negative control adeno-associated virus; AAV-Ambp, AMBP-overexpressing adeno-associated virus; AV, aortic valve; HE, hematoxylin and eosin; HCD, high cholesterol diet; qRT-PCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean.

[0024] Figure 3AMBP protects VIC osteoblast differentiation and calcification in vitro. (A) Four siRNAs specifically targeting AMBP were designed and transfected into VICs (valvular interstitial cells). The efficiency of AMBP knockdown was evaluated by qRT-PCR (n=6). (B) AMBP knockdown significantly enhanced OM-induced expression of RUNX2 and (B) AMBP at the mRNA level analyzed by qRT-PCR (n=8). VICs were transfected with SiR-NC or SiR-AMBP for 24 h and then cultured in DMEM or OM for 72 h. (C) AMBP knockdown significantly increased OM-induced expression of RUNX2 and OSTERIX at the protein level (n=6). VICs were transfected with SiR-NC or SiR-AMBP for 24 h and then cultured in DMEM or OM for 72 h. (D) Alizarin Red S staining showed that AMBP knockdown significantly enhanced OM-induced accumulation of calcium deposits and formation of calcified nodules (n=5). VICs were transfected with SiR-NC or SiR-AMBP for 24 h and then cultured in DMEM or OM for 7 d. (E) Representative immunoblot images and quantitative analysis of the effect of AMBP overexpression in VICs transfected with Ad-AMBP (n=6). (F) AMBP overexpression significantly inhibited the OM-induced expression of RUNX2 and OSTERIX at the mRNA level analyzed by qRT-PCR (n=6). VICs were transfected with Ad-Nc or ​​Ad-AMBP for 24 h and then cultured in DMEM or OM for 72 h. (G) AMBP overexpression significantly inhibited the OM-induced expression of RUNX2 and OSTERIX at the protein level (n=6). VICs were transfected with Ad-Nc or ​​Ad-AMBP for 24 h and then cultured in DMEM or OM for 72 h. (H) Alizarin Red S staining showed that AMBP overexpression could protect OM-induced calcium deposition accumulation and calcified nodule formation (n=5). VICs were transfected with Ad-Nc or ​​Ad-AMBP for 24 h and then cultured in DMEM or OM for 7 d. Values ​​are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: Ad-Nc, negative control adenovirus; Ad-AMBP, AMBP overexpressing adenovirus; DMEM, Dulbecco's modified Eagle's medium; OM, osteogenic medium; qRT-PCR, real-time quantitative polymerase chain reaction; SiR-NC, small interfering RNA negative control; SiR-AMBP-1, -2, -2, -3 and -4, small interfering RNAs targeting AMBP No.1, No.2, No.3 and No.4; VICs, valvular interstitial cells.

[0025] Figure 4AMBP gene knockdown increased the phosphorylation levels of ERK1 / 2 and JNK. (AB) RNA-Seq was performed on AVs in CAVD and non-CAVD groups. (A) Bubble plots show the KEGG pathway analysis of upregulated DEGs. The size of the bubble indicates the number of genes associated with each pathway, while the color gradient indicates the adjusted p-value (p.adj.), with more significant pathways indicated in red. The x-axis shows the gene ratio, and the y-axis lists the KEGG pathways. (B) Enrichment map of the MAPK signaling pathway from GSEA. (C) Representative immunoblot images of P-ERK1 / 2, ERK1 / 2, P-JNK, JNK, P-p38, and p38 after 24 h of transfection with SiR-AMBP or SiR-NC at different time points. (D) Quantitative analysis of the ratios of P-ERK1 / 2 to total ERK1 / 2 (D, n = 3), P-JNK to total JNK (E, n = 3), and P-p38 to total p38 (F, n = 3). Values ​​are expressed as mean ± SEM. *p < 0.05. Abbreviations: aortic valve, aortic valve; DEG, differentially expressed gene; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; OM, osteogenic medium; P-ERK1 / 2, phosphorylated ERK1 / 2; P-JNK, phosphorylated JNK; P-p38, phosphorylated p38; RNA-Seq, RNA sequencing; SEM, standard error; SiR-AMBP, small interfering RNA targeting AMBP; SiR-NC, small interfering RNA negative control; VICs, valvular interstitial cells.

[0026] Figure 5 AMBP competitively binds to the ZF domain of FHL3, promoting proteasomal degradation of activated MAPK pathway.

[0027] (A) Quantification of P-ERK1 / 2 and P-JNK expression in VICs transfected with Ad-AMBP or Ad-NC after pretreatment with the ubiquitin-proteasome specific inhibitor MG132 for 1 h (n=3). (B) Venn diagram showing the intersection of the BioGRID prediction dataset, the IntAct prediction dataset, and the DEGs found in this study. (C) Co-IP assay confirmed the interaction between AMBP and FHL3. HEK293T cells were transfected with Flag-tagged AMBP overexpression plasmid for 48 h. Cell lysates were subjected to Co-IP assay using Flag antibody, followed by immunoblotting of FHL3, CTSB, and PIK3CA to assess their interaction with AMBP. (D) The predicted interaction model between AMBP and FHL3 was generated using AlphaFold3. Crystal structure simulations visualized with PyMOL highlight the interaction interface between the two proteins. (E) Representative immunoblot images and quantification of P-ERK1 / 2 and P-JNK expression after transfection of SiR-FHL3 or SiR-NC for 1 h and OM induction for 72 h (n=3). (F) Co-IP assay confirmed the interaction between FHL3 and P-JNK and P-ERK1 / 2 when FHL3 was knocked down. VICs were transfected with SiR-FHL3 or SiR-NC for 48 h and then treated with OM for 10 min. Cell lysates were subjected to Co-IP assay using FHL3 antibody and then immunoblotted for P-JNK and P-ERK1 / 2 to evaluate their interaction with FHL3. (G) Co-IP assay confirmed the interaction between FHL3 and P-JNK and P-ERK1 / 2 with AMBP overexpression. Re-transfection with Ad-AMBP or Ad-NC for 48 h was performed and then treated with OM for 10 min. Cell lysates were Co-IPed with FHL3 antibody and then immunoblotted for P-JNK and P-ERK1 / 2 to assess their interaction with FHL3. (H) Schematic diagram of the major domains of FHL3, highlighting the five predicted functional domains: ZF domain (7-31AA) and four LIM zinc-binding domains (LIM140-92AA, LIM2101-153AA, LIM3162-212AA, and LIM4221-275AA). (I) Co-IP analysis showing the interaction between AMBP and full-length or truncated forms of FHL3. HEK293T cells were transfected with AMBP-Flag plasmid and full-length or truncated FHL3-HA plasmid for 48 h, and cell lysates were Co-IPed with Flag antibody and then immunoblotted with HA antibody to assess the interaction with AMBP-Flag. Values ​​are expressed as mean ± standard error.*p<0.05; **p<0.01. Abbreviations: Ad-Nc, negative control adenovirus; Ad-AMBP, AMBP overexpression adenovirus; AMBP-Flag, Flag-tagged AMBP overexpression plasmid; Co-IP, co-immunoprecipitation; OM, osteogenic medium; P-ERK1 / 2, phosphorylated ERK1 / 2; P-JNK, phosphorylated JNK; P-p38, phosphorylated p38; SEM, standard error; SiR-FHL3, small interfering RNA targeting FHL3; SiR-NC, small interfering RNA negative control; VICs, valvular interstitial cells.

[0028] Figure 6 AMBP protects osteoblast differentiation and calcification of VICs by inhibiting the MAPK pathway. (A) Representative immunoblot images and expression statistical analysis of RUNX2 and OSTERIX expression detected by Western blot after 1 h pretreatment with PD98059, a specific inhibitor of P-ERK1 / 2, and transfection with SiR-AMBP or SiR-NC (n=6). (B) Representative Alizarin Red S staining and quantitative statistical analysis of calcium deposition after 1 h pretreatment with PD98059 and 7 days of OMR induction with SiR-AMBP or SiR-NC (n=5). (C) Representative immunoblot images and expression statistical analysis of RUNX2 and OSTERIX expression detected by Western blot after 1 h pretreatment with SP600125, a specific inhibitor of P-JNK, and transfection with SiR-AMBP or SiR-NC (n=6). (D) Representative Alizarin Red S staining and quantitative statistical analysis of calcium deposition after 1 h of SP600125 pretreatment and 7 days of SiR-AMBP or SiR-NC transfection (n = 5). Values ​​are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide, solvent control as inhibitor; OM, osteogenic medium; SiR-AMBP, small interfering RNA targeting AMBP; SEM, standard error; SiR-NC, negative control small interfering RNA; VICs, valvular interstitial cells.

[0029] Figure 7 AMBP overexpression alleviates ApoE via MAPK pathway - / - Mouse AV calcification. (AE) ApoE - / - Mice were transfected with AAV-Ambp, fed HCD for 24 weeks, and pretreated with P-ERK1 / 2 or P-JNK specific activators, bortezomib or anisomycin, respectively. (A) qRT-PCR was used to quantify the expression of Runx2 and Osterix in AVs (n=6). (B) Representative HE images and ApoE - / -Quantitative analysis of mouse aortic valve thickening (n=4, scale bar=0.05 mm). (C) Representative Masson staining images and ApoE - / - Quantitative analysis of aortic valve fibrosis in mice (n = 4, scale bar = 0.2 mm). (D) Representative images of Alizarin Red S (black arrows indicate calcified lesions) and Von Kossa (red arrows indicate calcified lesions) staining and quantitative analysis of aortic valve calcification (n = 4, scale bar = 200 μm). (E) Representative images of aortic valve pulsed Doppler blood flow profiles and quantitative analysis of transvalvular peak jet velocity and aortic valve peak pressure assessed by echocardiography (n = 6). Values ​​are expressed as mean ± SEM. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; *****p < 0.0001. Abbreviations: AAV-Ambp, Ambp overexpressing adeno-associated virus; AVs, aortic valve; ND, normal diet; HCD, high cholesterol diet; SEM, standard error.

[0030] Figure 8 .Schematic diagram of the mechanism by which AMBP antagonizes aortic valve calcification by inhibiting FHL3-mediated ERK1 / 2 and JNK pathways.

[0031] Fig. 9 .GO and KEGG enrichment analysis of differentially expressed genes (DEGs), (A) GO enrichment analysis of DEGs, showing the top 30 enriched terms; (B) KEGG pathway analysis of DEGs. Shows the top 30 enriched pathways. DEGs, differentially expressed genes; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

[0032] Fig.10 .PPI network construction and MCODE analysis diagram, (A) PPI network constructed. Red genes indicate up-regulated genes, blue genes indicate down-regulated genes; (B) The top seven significant modules obtained using MCODE; (C) Venn diagram verifies the overlap of core genes in the five algorithms. MCODE, molecular complex detection; PPI, protein-protein interaction.

[0033] Fig.11 .ApoE detection - / - Serum glucose and lipid levels in mice transfected with AAV-Ambp or AAV-Nc after feeding a high cholesterol diet (HCD) for 24 weeks were affected by ApoE - / -Glucose (A), total cholesterol (B), triglycerides (C), high-density lipoprotein cholesterol (D), and low-density lipoprotein cholesterol (E) in mouse serum were analyzed (n = 6). ns, no significant difference; AAV-Nc, negative control adeno-associated virus; AAV-Ambp, adeno-associated virus overexpressing Ambp; HCD, high cholesterol diet.

[0034] Fig.12 . Validation of knockdown efficiency of two siRNAs specifically targeting FHL3. (A) Two siRNAs specifically targeting FHL3 were designed and transfected into VICs, and the knockdown efficiency of FHL3 was evaluated by Western blot (n=5). DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] AMBP, alpha-1-microglobulin / bikunin precursor;

[0037] Aortic valve; CAVD, calcific aortic valve disease;

[0038] ERK, extracellular signal-regulated kinase;

[0039] JNK, c-Junn terminal kinase.

[0040] The purpose of this study is to identify the key pathogenic genes of CAVD and elucidate the mechanisms that may guide targeted treatment of CAVD.

[0041] method:

[0042] ApoE knockout mice were established, and a CAVD model was constructed by feeding a high cholesterol diet for 24 weeks. AMBP overexpression was induced by adeno-associated virus. Aortic valve function was evaluated by RNA sequencing, qRT-PCR, immunoblotting, immunofluorescence, histopathology, and echocardiography. The interaction mechanism between AMBP and FHL3 was explored by bioinformatics analysis, co-immunoprecipitation, and AlphaFold3-based crystal structure simulation.

[0043] Results: RNA sequencing results showed that AMBP is a key regulator of CAVD. AMBP overexpression significantly reduced ApoE expression induced by high cholesterol diet in vivo. - / -Aortic valve (AV) calcification and fibrosis in mice. In vitro, downregulation of AMBP increased the expression of osteogenic markers RUNX2 and OSTERIX and promoted calcium deposition in valvular interstitial cells induced by osteogenic medium (OM), while overexpression of AMBP reversed these effects. Mechanistically, AMBP inhibited OM-induced phosphorylation of ERK1 / 2 (P-ERK1 / 2) and JNK (P-JNK) by competitively binding to the zinc finger domain of FHL3. This interaction abolished the protective role of FHL3 in preventing ubiquitin-proteasome-mediated degradation of P-ERK1 / 2 and P-JNK. In vitro and in vivo experiments demonstrated that P-ERK1 / 2 and P-JNK inhibitors and agonists confirmed that the protective effect of AMBP against CAVD was mediated through P-ERK1 / 2 and P-JNK.

[0044] Conclusions: AMBP protects valvular interstitial cells from osteoblastic differentiation and calcium deposit accumulation, thereby attenuating AV calcification. This study sheds further light on the development of CAVD and potential new treatments.

[0045] Patients and sample collection

[0046] We included patients (aged 50-75 years) who underwent aortic valve replacement and had calcified aortic valve tissue collected. Non-calcified aortic valve tissue was collected from patients (also aged 50-75 years) who underwent heart transplantation (recipient heart) or aortic valvectomy for aortic dissection as controls (non-CAVD). Exclusion criteria were defined as: rheumatic heart disease, moderate or severe aortic insufficiency, chronic kidney disease, estimated glomerular filtration rate ≤ 30 mL / min / 1.73 m 2 , intestinal diseases (Crohn's disease and inflammatory bowel disease), autoimmune diseases, antibiotic or probiotic treatment within 1 month due to infection, or lack of clinical data. Demographic characteristics, medical history, physical examination results, major laboratory test results, and cardiac imaging data of all patients were collected. This study conformed to the principles of the Declaration of Helsinki and was approved by the Research Ethics Committee of Qilu Hospital of Shandong University (Ethics Approval No.: KYLL-202208-003-1). Written informed consent was obtained from all patients.

[0047] RNA-seq data generation and bioinformatics analysis

[0048] Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and its integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The concentration and purity of RNA were measured using a 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and a NanoDrop 1 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared from stranded mRNA and sequenced using a NovaSeq 6000 (San Diego, CA, USA).

[0049] For bioinformatics analysis, sequencing data were processed using Fastp to filter out low-quality reads and adapters. Clean reads were aligned to the human reference genome (GRCh38.102) using Hisat2 (version 2.0.5), and the resulting SAM files were converted to BAM format and sorted using SAMtools (version 1.3.1). StringTie was used for fragment counting (https: / / ccb.jhu.edu / software / stringtie / ), TMM for normalization, and EdgeR for differential expression analysis. Heatmap and ggplot2 packages in R were used for visualization, and DEGs were defined as |log2(FC)|>1 and q-value (FDR-adjusted P-value)<0.05. They were further analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment techniques. Protein-protein interaction (PPI) networks were constructed using STRING and visualized using Cytoscape (version 3.9.1) and hub genes were identified by CytoHubba. RNA-seq data are available under GEO accession number GSE235995.

[0050] Isolation of total RNA from tissues and cells and qRT-PCR

[0051] Total RNA was isolated from aortic valve tissues and cells using TRIzol reagent (Invitrogen) and reverse transcribed using PrimeScript RT reagent kit (Takara Biomedical Technology, Beijing, China). qRT-PCR amplification was performed using SYBR PCR mix and specific primers (as shown in Table S1) in a Bio-Rad CFX96TM Real-Time PCR Detection System (Bio-Rad Laboratories, Bio-Rad Laboratories, CA, USA).

[0052] Table S1. Specific primer sequences used for qRT-PCR amplification

[0053]

[0054]

[0055] Isolation of primary VIC cells, cell culture and transfection

[0056] Primary human VICs were isolated from patients without CAVD who received heart transplantation (recipient hearts) or from patients who had aortic valves removed for aortic dissection. The clinical characteristics of the patients used for cell isolation are listed in Table S2. Materials VICs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Osteoblast differentiation was stimulated using osteogenic medium (OM). Small interfering RNAs (siRNAs) targeting AMBP and FHL3 (Cat. No. 13778150; Thermo Fisher Scientific) or AMBP-overexpressing adenovirus (BIOSUN, Jinan, China) were transfected using Lipofectamine RNAi MAX Transfection Reagent.

[0057] Table S2. Clinical characteristics of patients used for cell extraction

[0058]

[0059]

[0060] Values ​​are expressed as mean ± standard error (SEM) or median (interquartile range). ALT, alanine aminotransferase; AST, aspartate aminotransferase; CR, creatinine; DBIL, direct bilirubin; HDL-C, high-density lipoprotein cholesterol; IBIL, indirect bilirubin; LDL-C, low-density lipoprotein cholesterol; LVEF, left ventricular ejection fraction; SEM, standard error; TG, triglyceride; UA, uric acid.

[0061] For mechanistic studies, VICs were treated with P-ERK1 / 2 inhibitor (50 μM; Catalog No. PD98059; Topology Science, Shanghai, China), P-JNK inhibitor (10 μM; Catalog No. SP600125; Topology Science), or proteasome inhibitor MG132 (10 μM; Catalog No. HY-13259; MCE, Shanghai, China) for 1 h and then transfected with siRNA or adenovirus. For PPI experiments, HEK293T cells were transfected with plasmids encoding C-terminal FLAG-tagged AMBP and C-terminal HA-tagged GFP using Lipofectamine 3000 (Catalog No. L3000015; Thermo Fisher Scientific) following the manufacturer's protocol.

[0062] Protein extraction and western blot analysis

[0063] VICs were lysed in RIPA buffer (Sigma-Aldrich, USA) containing 1X protease inhibitors (Cat. No. 04693132001; Roche, Indianapolis, IN). The extracted proteins were separated by 4-10% gradient Bis-Trissds-gel (Bio-Rad, Hercules, CA, USA) and then transferred to nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were incubated with 5% skim milk at room temperature (23-27°C) for 1 hour and then incubated with primary antibodies overnight at 4°C.

[0064] The next day, the cell membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cat. No.: ab6721 (anti-rabbit); ab6728 (anti-mouse); USA) at room temperature (23-27°C) for 1 h and visualized using an ECL Western Blot Detection Kit. The grayscale of the immunoblot bands was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to GAPDH or -actin.

[0065] Alizarin Red S staining of VICs

[0066] The treated primary VICs were washed with phosphate-buffered saline for 3 min, fixed in 4% paraformaldehyde for 30 min, and then treated with 0.2% Alizarin Red solution (Cat. No. C0148S; Sewell, Wuhan, China) for 1 h. Excess dye was removed with double distilled water. Alizarin Red S staining was observed and photographed with an inverted microscope (Olympus, Tokyo, Japan). Representative images of each group were randomly selected.

[0067] Co-immunoprecipitation

[0068] 48 hours after plasmid transfection, cells were collected and lysed in non-denaturing lysis buffer (Cat. No.: P0013; Bio-Tech) for 30 min. Protein A / G magnetic beads were incubated with immunoprecipitation grade antibodies at room temperature for 1 h, and then cell lysis buffer was added, followed by rotation and incubation at 4°C overnight. The next day, beads were collected and supernatant was discarded. After washing four times with PBS containing 0.5% Tween-20, SDS-PAGE loading buffer was added, and samples were heated at 95°C for 5 min. The collected supernatant was used for immunoblot analysis.

[0069] Immunofluorescence staining

[0070] Human aortic valve tissue sections were dewaxed, antigen retrieved, and treated with 0.1% TritonX-100 for 10 min. The sections were then incubated with 2.5% normal goat serum at room temperature for 30 min, and then with primary antibodies against AMBP (Cat. No. ER1803-35; HUABIO, Hangzhou, China) and VIM (Cat. No. 5741; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The next day, the sections were incubated with Alexa Fluor 594 (Cat. No. ab150120; Abcam) and 488 (Cat. No. ab150081; Abcam) secondary antibodies (1:200) at 37°C in the dark for 1 hour. Cell nuclei were stained with DAPI (Cat. No. ab104139; Abcam). Immunofluorescence staining was performed using a fluorescence microscope (Olympus).

[0071] Animal experiments

[0072] All animal procedures were approved by the Institutional Review Board of Qilu Hospital of Shandong University and complied with Regulation 2010 / 63 / EU for the protection of animals. - / - Mice (C57BL / 6J background) were purchased from Jicui Pharmaceutical Co., Ltd. (Jiangsu, China) and maintained in a pathogen-free, temperature-controlled environment with a 12:12 h light-dark cycle. Mice were injected with adeno-associated virus (2 × 10^11 vg / mouse; CMV promoter; Gene Pharmaceuticals, Shanghai, China) overexpressing AMBP via tail vein for 2 weeks and then fed a 0.2% high cholesterol diet for 24 weeks to induce aortic valve calcification.

[0073] In the in vivo mechanistic experiment, mice were injected with adeno-associated virus (AAV) Ambp and pretreated with ERK activator bortezomib (Cat. No. S1013; Selleck, Shanghai, China) and JNK activator anisomycin (Cat. No. S7409; Selleck) before initiating HCD. At the end of the experiment, echocardiography was performed using an 18-38 MHz phased array transducer (MS400) and VisualSonic VeVo2100 imaging system (Toronto, Canada) to measure transvalvular peak jet velocity and aortic valve peak pressure.

[0074] Mice were anesthetized with 2% isoflurane and placed on a heated platform at 37 ± 1 °C. After imaging, mice were euthanized with a lethal dose of sodium pentobarbital (100 mg / kg), and aortic valve tissue and blood were collected for analysis. OCT-embedded frozen sections (4 μm) were used for histological examination and stained with Masson, Alizarin Red S, and Von Kossa. The stained sections were photographed under a light microscope (Olympus). All animals were treated in accordance with the established animal care guidelines approved by the Research Ethics Committee of Qilu Hospital of Shandong University (approval number: KYLL-2023(ZM)-360).

[0075] Molecular modeling and docking

[0076] The AMBP and FHL3 protein sequences were derived from the UniProt database. The sequences were submitted to the AlphaFold server for protein structure prediction and interaction analysis. The predicted interaction model was visualized using PyMOL (PyMOL molecular graphics system), which highlights the polar interactions between proteins to illustrate the key binding sites.

[0077] Statistical analysis

[0078] Data were analyzed using GraphPad Prism 9 software (San Diego, CA, USA). Continuous variables were expressed as mean ± standard error of the mean (SEM) when they were normally distributed; otherwise, they were expressed as median and interquartile range. Normality was tested using the Shapiro–Wilk normality test. For normally distributed data, unpaired two-tailed Student’s t-test was used to determine statistically significant differences between two groups. One-way ANOVA followed by Bonferroni multiple comparison test (mixed model with different numbers of replicates per condition) was used to determine statistical differences between multiple groups with univariate and normal distribution. Two-way ANOVA followed by Bonferroni multiple comparison test was used to compare multiple groups with multiple variables. For non-normally distributed data, the Kruskal–Wallis test, a nonparametric statistic, was performed for multiple comparisons, followed by Dunn’s post hoc test. Statistical significance was set at p < 0.05.

[0079] result

[0080] AMBP is increased in calcified AVs of CAVD patients and mice

[0081] Calcified human aortic valve tissue was collected from 5 patients (4 males) with severe AVS (aortic stenosis). Control aortic valve tissue (non-CAVD) was collected from 1 patient (female) who underwent heart transplantation for refractory dilated cardiomyopathy and 3 patients (males) who underwent aortic valvulectomy for aortic dissection. Patients in the non-CAVD group were younger than those in the CAVD group (51.75±4.64 years vs. 64.80±1.69 years, P<0.05). There were no significant differences between the two groups in other demographic characteristics, non-cardiac history, routine laboratory indices, or cardiac function (Table S3).

[0082] Table S3. Clinical characteristics of CAVD and non-CAVD cases by aortic valve tissue RNA sequencing

[0083]

[0084]

[0085] Values ​​are expressed as mean ± standard error (SEM) or median (interquartile range). ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAVD, calcific aortic valve disease; CR, creatinine; DBIL, direct bilirubin; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; IBIL, indirect bilirubin; LDL-C, low-density lipoprotein cholesterol; LVEF, left ventricular ejection fraction; NA, not applicable; SBP, systolic blood pressure; SEM, standard error; TG, triglycerides; UA, uric acid.

[0086] RNA-seq was performed on AVs in the CAVD and non-CAVD groups to identify differentially expressed genes (DEGs). A total of 870 DEGs were identified, including 525 up-regulated genes and 345 down-regulated genes (|log2(FC)|>1 and q<0.05)( Figure 1 A, 1B). The first 50 deg are shown in Table S4.

[0087] Table S4. List of the top 50 differentially expressed genes

[0088]

[0089]

[0090] GO enrichment analysis showed that DEGs were enriched in 582 GO terms (q≤0.05), including 480 biological processes (BP), 60 cellular components (CC), and 42 molecular functions (MF). The top 30 GO terms were mainly related to lipid composition, metabolism, and immune cell processes ( Fig. 9KEGG pathway analysis showed that the DEGs were related to extracellular matrix (ECM) interaction, mineral absorption, and cholesterol metabolism (e.g. Fig. 9 To explore DEG interactions, we constructed a PPI network using the STRING database, which consisted of 229 nodes and 328 edges with an interaction score threshold of 0.9 (as shown in Figure 2B). Fig.10 The network included 146 up-regulated genes and 79 down-regulated genes in the CAVD group. Module analysis was performed using the molecular complex detection (MCODE), and 17 significant modules were identified (Table S5).

[0091] Table S5. List of 17 obvious modules obtained from MCODE analysis

[0092]

[0093]

[0094] The first seven modules are shown in Fig.10 In B, five algorithms (Dedare, DMNC, EPC, MCC, and MNC) were applied to identify key hub genes using the CytoHubba plug-in. The top 30 hub genes were identified (as shown in Table S6), and seven overlapping hub genes were confirmed using the Venn diagram: AMBP, FGG, FGA, SERPINC1, APOA2, APOB, and ACAN ( Figure 1 C). The expression of these 7 key hub genes in the two groups of av was further verified by qRT-PCR. Consistent with the RNA-seq data, all 7 genes were significantly upregulated in the CAVD group compared with the non-CAVD group, indicating their potential role in the pathogenesis of CAVD ( Figure 1 D).

[0095] Table S6. List of the top 30 core genes obtained by the five algorithms of Cytohubba

[0096]

[0097]

[0098] According to gene function analysis and literature search, ACAN, FGA, FGG, and SERPINC1 are mainly involved in the expression or degradation of the encoded ECM proteins, which are mainly involved in the final stage of aortic valve calcification. APOA2 and APOB encode apolipoproteins A2 and B, respectively, which are involved in lipid metabolism and the initial stage of CAVD pathogenesis. Unlike other studies, the role of AMBP, plasma glycoprotein alpha-1-microglobulin (A1M), and bikunin precursor protein in the pathogenesis of CAVD remains a mystery and has not been reported.

[0099] The AMBP gene encodes the plasma glycoprotein A1M and bikunin precursor protein. AMBP is hydrolyzed into two proteins with different functions: A1M protein and bikunin protein. A1M is an antioxidant and tissue cleaning protein with reductase, heme and free radical binding properties and plays a role in regulating inflammation. However, bikunin is a structural component of the ECM. Therefore, AMBP may be an important regulatory factor involved in the occurrence and development of CAVD.

[0100] Immunofluorescence staining showed that AMBP co-localized with vimentin (a specific marker protein of VICs). Figure 1 E). These findings strongly suggest that VICs may be the main cellular source of elevated AMBP expression in calcified aortic valve tissue. Quantitative analysis of immunofluorescence staining showed that the expression of AMBP in AV tissue (aortic valve tissue) of CAVD patients was significantly higher than that in non-CAVD patients ( Figure 1 F). A CAVD model was established in ApoE (apolipoprotein E) knockout mice by feeding them HCD (high cholesterol diet) for 24 weeks to induce aortic valve calcification ( Figure 1 G). Immunohistochemical analysis showed that the expression of AMBP in the AV tissue of HCD mice was significantly upregulated compared with that of normal diet (ND) mice ( Figure 1 H). We investigated the temporal expression pattern of AMBP in OM-induced VICs in vitro. The expression of AMBP was gradually upregulated with the prolonged OM treatment. Specifically, statistical analysis showed that the expression of AMBP was significantly upregulated at the 48 h and 72 h time points compared with the baseline (0 h) control group ( Figure 1 I). These results indicate that AMBP is upregulated in calcified aortic valves in vivo and in VICs induced by osteogenic conditions in vitro.

[0101] AMBP overexpression attenuates HCD-induced aortic valve calcification in ApoE mice

[0102] To investigate the role and therapeutic potential of AMBP overexpression in an animal model of AV calcification, 6-week-old ApoE- / - mice (ApoE knockout mice) were injected with AAV (adeno-associated virus) overexpressing Ambp via the tail vein for 2 weeks. Subsequently, the mice were fed HCD (high cholesterol diet) for 24 weeks to induce aortic valve calcification ( Figure 2 A). qRT-PCR was used to verify the efficiency of AMBP overexpression in AV 2 weeks after AAV injection ( Figure 2 B). At the end of the modeling period, qRT-PCR analysis of osteoblast differentiation marker genes in the AV lobule showed that overexpression of Ambp significantly inhibited the HCD-induced expression of Runx2 and Osterix ( Figure 2 C). HE staining showed that Ambp overexpression significantly inhibited the HCD-induced AV leaflet thickness ( Figure 2 D). Masson trichrome staining showed that Ambp overexpression significantly inhibited HCD-induced lobular fibrosis ( Figure 2 E). Alizarin Red S and Von Kossa staining showed that Ambp overexpression significantly inhibited HCD-induced AV leaflet calcification ( Figure 2 F). Echocardiographic assessment showed that Ambp overexpression significantly reduced transvalvular peak ejection velocity and aortic valve peak pressure in HCD-fed ApoE- / - mice ( Figure 2 G). Further, no differences in serum glucose and lipid levels, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol, and LDL-C, were observed between AAV-Nc and AAV-Ambp mice fed HCD, suggesting that the protective effect of Ambp against CAVD is independent of glucose and lipid metabolism (e.g. Fig.11 In summary, Ambp overexpression alleviates HCD-induced A compartment calcification in ApoE- / - mice.

[0103] AMBP protects osteoblastic differentiation and calcification of valvular interstitial cells (VICs) in vitro

[0104] To investigate the effect of AMBP on VIC calcification in vitro, we used four siRNAs targeting AMBP for knockdown. The second siRNA showed the highest knockdown efficiency in primary VICs, significantly reducing AMBP expression and validating its use in further functional studies ( Figure 3 A). qRT-PCR results showed that AMBP knockout significantly increased the expression of osteogenic marker genes RUNX2 and OSTERIX under OM induction, indicating that AMBP inhibited osteoblast differentiation ( Figure 3 B). The results obtained using western blot analysis were consistent ( Figure 3C). Alizarin red S staining further showed that OM significantly induced calcium deposition accumulation and calcified nodule formation. AMBP knockdown significantly enhanced OM-induced calcium deposition and nodule formation ( Figure 3 D). These results indicate that AMBP knockdown promoted osteoblast differentiation and calcium deposition in VIC under OM conditions.

[0105] In addition, primary VICs were transfected with AMBP-overexpressing adenovirus to elucidate the effect of AMBP on VIC calcification in vitro. The expression of AMBP was significantly increased by immunoblotting, confirming the successful overexpression of AMBP, which supports its application in subsequent functional studies ( Figure 3 E). qRT-PCR results showed that AMBP overexpression significantly reduced the expression of osteogenic markers RUNX2 and OSTERIX under OM induction ( Figure 3 F). Western blot analysis confirmed these findings ( Figure 3 G). Alizarin Red S staining further showed that AMBP overexpression significantly reduced OM-induced calcium deposition and nodule formation ( Figure 3 H). These results suggest that AMBP overexpression inhibits the differentiation of VICs into an osteoblastic phenotype and plays a key role in reducing calcium deposition under OM conditions.

[0106] AMBP knockdown activates the mitogen-activated protein kinase (MAPK) signaling pathway in VICs

[0107] To investigate the mechanism of AMBP in protecting osteoblast differentiation and calcification, KEGG pathway analysis was performed using RNA-seq data of differentially upregulated genes in CAVD patients compared with controls to identify the most significantly enriched pathways in the MAPK signaling pathway in the CAVD group ( Figure 4 ). Similarly, previous studies have shown that the MAPK pathway mediates osteoblast differentiation and RUNX2 expression in osteoblast-like cell lines MG 63 and COS7. Gene set enrichment analysis (GSEA) of the MAPK signaling pathway was performed to investigate its role on a genome-wide scale. According to the GSEA results, the MAPK signaling pathway was significantly enriched in the gene expression data of CAVD patients ( Figure 4 B). This demonstrates a strong association between the MAPK pathway and CAVD development, further supporting its potential role in the pathophysiology of this disease.

[0108] The effects of AMBP on three key members of the MAPK pathway: p38, JNK, and ERK1 / 2 were studied. The results showed that OM exposure significantly induced the phosphorylation of ERK1 / 2 (P-ERK1 / 2), JNK (P-JNK), and p38 (P-p38) in a time-dependent manner. Specifically, the levels of P-ERK1 / 2, P-JNK, and P-p38 peaked at 10 min after OM treatment and gradually decreased at 30 and 60 min, indicating that the MAPK pathway was activated under OM stimulation ( Figure 4 C). In addition, OM treatment significantly reduced total ERK1 / 2 levels, increased total p38 levels, and had no effect on total JNK levels ( Figure 4 C). AMBP gene knockout significantly increased the ratio of P-ERK1 / 2 to total ERK1 / 2 ( Figure 4 D) and the ratio of P-JNK to total JNK ( Figure 4 E), while the ratio of P-p38 to total p38 did not change significantly ( Figure 4 F). These results indicate that AMBP significantly inhibited the phosphorylation of ERK1 / 2 and JNK in the MAPK signaling pathway. The regulatory effect of AMBP suggests that it affects osteoblast differentiation through the MAPK pathway in VICs, suggesting that MAPK may be a key pathway in this cellular process.

[0109] AMBP competitively binds to the ZF domain of FHL3 and promotes proteasomal degradation of activated MAPK pathway

[0110] Intracellular protein degradation is a fundamental process for maintaining cellular homeostasis and is mainly controlled by the ubiquitin-proteasome system (UPS) and autophagy. The UPS is responsible for degrading more than 80% of intracellular proteins. Recent studies have shown that the UPS pathway is involved in the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and that ubiquitination plays a role in ERK1 / 2 degradation in HEK293T cells. In this study, we aimed to investigate whether the inhibition of JNK and ERK1 / 2 phosphorylation by AMBP is mediated by the UPS. To further explore the effect of AMBP on the expression of P-JNK and P-ERK1 / 2 in VICs, the proteasome inhibitor MG132 was used to block UPS-mediated degradation. Western blot analysis showed that the inhibitory effect of AMBP on the expression of P-JNK and P-ERK1 / 2 was significantly reversed after pretreatment with MG132 ( Figure 5 A), indicating that the inhibition of AMBP by P-JNK and P-ERK1 / 2 is UPS-mediated.

[0111] To investigate the specific mechanism by which AMBP exerts this effect, we used the PPI databases IntAct and BioGRID to identify proteins that may interact with AMBP and cross-referenced them with the DEGs in our study ( Figure 5 B). We identified three potential AMBP interactors: FHL3, CTSB, and PIK3CA. Co-immunoprecipitation (Co-IP) assays confirmed that AMBP significantly interacted with FHL3, but not with CTSB or PIK3CA, in HEK293T cells ( Figure 5 C). The crystal structure and interaction interface between AMBP and FHL3 were simulated using AlphaFold3 ( Figure 5 D).

[0112] Next, we explored how FHL3 mediates AMBP ubiquitination and P-ERK1 / 2 ubiquitination and degradation. Previous studies have reported that FHL3 gene downregulation can significantly inhibit the phosphorylation of MAPK pathway components p38, ERK1 / 2, and JNK in gastric cancer cell lines, and FHL3 can prevent the ubiquitination and degradation of substrate proteins. In this study, we constructed siRNA targeting FHL3 and selected siRNA No. 2 with the highest knockdown efficiency for subsequent experiments (such as Fig.12 Western blot results showed that knockdown of FHL3 significantly reduced the expression of P-JNK and P-ERK1 / 2 in OM-induced VICs, while MG132 pretreatment reversed this effect ( Figure 5 E). Co-IP analysis further demonstrated that FHL3 directly binds to P-JNK and P-ERK1 / 2, and this interaction is significantly attenuated after FHL3 knockdown in OM-induced VIC ( Figure 5 F). These findings suggest that in OM-activated VICs, FHL3 binds to P-JNK and P-ERK1 / 2 and protects them from UPS-mediated degradation. We then investigated how AMBP mediates the degradation of P-JNK and P-ERK1 / 2 through its interaction with FHL3. Co-IP revealed that AMBP overexpression significantly reduced the binding of FHL3 to P-JNK and P-ERK1 / 2 in OM-induced VICs (valvular interstitial cells). Figure 5G). This suggests that AMBP competitively binds to FHL3, thereby promoting the ubiquitination and degradation of P-JNK and P-ERK1 / 2 in OM-activated VICs. To elucidate the key domains required for the interaction between FHL3 and AMBP, we analyzed the main domains of FHL3 and identified five predicted functional domains: a zinc finger domain (ZF, 7-31AA) and four LIM zinc-binding domains (LIM1, 40-92AA, LIM2, 101-153AA, LIM3, 162-212AA; and LIM4, 221-275AA) ( Figure 5 H).

[0113] Plasmids encoding full-length FHL3 and five truncated fragments fused to a C-terminal HA tag were constructed and co-transfected with a flag-tagged AMBP overexpression plasmid into HEK293T cells. FHL3-HA was pulled down using an anti-Flag antibody, and Co-IP results showed that the loss of the ZF domain abolished the interaction between FHL3 and AMBP, while full-length FHL3 and truncated fragments lacking the LIM1, LIM2, LIM3, and LIM4 domains retained the ability to bind to AMBP ( Figure 5 I). These results suggest that the ZF domain of FHL3 is critical for its interaction with AMBP. Taken together, these findings suggest that AMBP disrupts the protective effect of FHL3 on P-JNK and P-ERK1 / 2 by competitively binding to the ZF domain of FHL3, preventing their degradation through the UPS. This leads to enhanced ubiquitination and subsequent degradation of P-JNK and P-ERK1 / 2 in OM-activated VIC cells. These results reveal a novel regulatory mechanism by which AMBP modulates MAPK signaling in calcific aortic valve disease.

[0114] AMBP protects osteoblast differentiation and calcification of VICs by inhibiting the MAPK pathway

[0115] Using specific P-ERK1 / 2 and p-JNK inhibitors, we investigated whether P-ERK1 / 2 and p-JNK mediate the protective effect of AMBP on VICs differentiation into osteoblasts. Immunoblotting analysis showed that knockdown of AMBP by siRNA led to a significant upregulation of RUNX2 and OSTERIX expression. However, pretreatment with the P-ERK1 / 2 specific inhibitor PD98059 significantly attenuated this increase ( Figure 6 A). Alizarin Red S staining showed that AMBP knockdown significantly increased the accumulation of calcium deposits, while PD98059 pretreatment significantly reduced the accumulation of calcium deposits ( Figure 6B). Similarly, pretreatment with SP600125, a specific inhibitor of p-JNK, significantly inhibited the increased expression of RUNX2 and OSTERIX caused by downregulation of AMBP ( Figure 6 C). Alizarin Red S staining also showed that SP600125 pretreatment significantly reduced the enhanced calcium deposition after AMBP knockdown ( Figure 6 D). These results suggest that P-ERK1 / 2 and p-JNK are key mediators of the protective effects of AMBP on osteoblast differentiation and calcification.

[0116] AMBP overexpression alleviates AV calcification via the MAPK pathway in ApoE- / - mice

[0117] To further explore whether AMBP reduces aortic valve calcification through the MAPK pathway in vivo, ApoE- / - mice were injected with type A overexpressing adeno-associated virus through the tail vein and treated with ERK1 / 2 specific agonist bortezomib or JNK agonist anisomycin by intraperitoneal injection. Mice were fed 0.2% HCD for 24 weeks to induce aortic valve calcification. qRT-PCR results in aortic valve tissue showed that after pretreatment with bortezomib and isomycin, the inhibitory effect of Ambp overexpression on calcification marker genes Runx2 and Osterix was significantly weakened ( Figure 7 A). Histological examination with HE staining showed that pretreatment with bortezomib and anisomycin significantly reversed the protective effect of AMBP on lobular thickness ( Figure 7 B). Similarly, Masson's Figure 7 C), Alizarin Red S and VonKossa staining ( Figure 7 D) showed that the protective effect of AMBP on AVs fibrosis and calcification was attenuated after pretreatment with both activators. Echocardiography showed that the beneficial effects of AMBP overexpression on transvalvular peak ejection velocity and AV peak pressure were abolished after pretreatment with bortezomib and isomycin, but AV peak pressure was abolished in the isomycin group ( Figure 7 E). These results suggest that the protective effect of AMBP against AV calcification is mediated through the MAPK pathway in Ap- / - mice.

[0118] discuss

[0119] In this study, RNA-seq analysis identified seven key hub genes involved in the pathogenesis of CAVD. Among them, AMBP emerged as an important regulator that significantly attenuated aortic valve calcification in ApoE- / - mice. In vivo experiments confirmed that overexpression of AMBP significantly reduced aortic valve calcification and leaflet fibrosis, thereby improving valvular function. In vitro complementary studies showed that AMBP knockdown significantly upregulated the expression of osteogenic markers RUNX2 and OSTERIX, while increasing calcium deposition in the VIC. In contrast, AMBP overexpression had the opposite effect. Our in vitro studies showed that AMBP exerted its protective effect by inhibiting the phosphorylation of ERK1 / 2 and JNK pathways, thereby reducing the expression of RUNX2 and OSTERIX and reducing calcium deposition in the VIC. In vivo experiments confirmed these findings and further demonstrated that the protective effect of AMBP against aortic valve calcification in ApoE- / - mice was mediated through the P-ERK1 / 2 and P-JNK pathways. Mechanistically, AMBP inhibits the phosphorylation of ERK1 / 2 and JNK pathways by competitively binding to the ZF domain of FHL3, thereby destroying the protective effect of FHL3 on P-JNK and P-ERK1 / 2 and promoting their ubiquitin-proteasome-mediated degradation. These findings provide important insights into the molecular mechanism by which AMBP affects CAVD and suggest its potential as a therapeutic target.

[0120] Our comprehensive transcriptome sequencing analysis of AV tissue from patients with CAVD provided several key insights. First, our results confirmed the dysregulation of genes associated with lipid composition and metabolism, confirming previous studies highlighting the key role of lipid metabolism in the initiation and progression of CAVD. Specifically, the alterations in apoB, apoA1, apo(a), eNOS, and NADPH oxidase levels observed in calcified AV confirm the involvement of lipoprotein dysregulation and oxidative stress in promoting inflammation and immune cell adhesion. Furthermore, the enrichment of GOterms related to nitric oxide synthase and immune cell processes emphasizes the active inflammatory nature of CAVD and highlights the important contribution of immune cells to disease pathogenesis. Second, our KEGG pathway enrichment analysis revealed the significant involvement of ECM receptor interactions and mineral absorption pathways, highlighting the key role of abnormal ECM remodeling in CAVD. ECM components are essential to coordinate the pathological processes of fibrosis, chronic inflammation, and lobular calcification. These findings reinforce the concept that CAVD is a complex pathophysiological process involving intricate interactions between VICs, valvular endothelial cells (VECs), inflammatory cells, and the ECM. Although hypercholesterolemia is a well-established risk factor for CAVD, several randomized clinical trials have demonstrated that lipid-lowering therapies, such as statins, have limited efficacy in slowing aortic valve calcification. This highlights the significant challenges in the management of CAVD and underscores the need for alternative treatment strategies. Therefore, further research is urgently needed to bridge this treatment gap and develop effective interventions for CAVD.

[0121] In this study, we identified seven key hub genes associated with CAVD: AMBP, FGG, FGA, SERPINC1, APOA2, APOB, and ACAN. Previous studies have shown that ACAN, FGA, FGG, and SERPINC1 are essential for regulating ECM proteins, thereby promoting ECM remodeling and degradation, which are fundamental processes in the pathogenesis of CAVD. In addition, APOA2 and APOB are integral to lipid metabolism and key players in the progression of CAVD. A1M is derived from AMBP and plays a role in regulating oxidative stress and inflammatory responses, both of which play a key role in the development and progression of CAVD. In addition, another AMBP product, bikunin, serves as a structural component of the ECM, further emphasizing the multifaceted role of AMBP in maintaining ECM integrity and regulating inflammatory responses. These findings highlight the complex interplay between lipid metabolism, ECM remodeling, and inflammatory processes in CAVD. Understanding the specific roles and interactions of these hub genes will provide valuable insights into the molecular mechanisms of CAVD and highlight potential therapeutic targets for the management of this debilitating disease.

[0122] Among the identified hub genes, AMBP has emerged as a potential key regulator of the pathogenesis of CAVD. Previous studies have shown that AMBP is upregulated in response to oxidative stress, suggesting that it plays a role in antioxidant defense mechanisms. Consistent with these observations, our study showed that the expression of AMBP in VICs was significantly increased in aortic valve tissue from patients with CAVD. In addition, our in vitro experiments showed that the expression of AMBP in VICs was time-dependently upregulated after osteogenic stimulation. Together, these findings suggest that AMBP plays a key role in the regulation of CAVD, possibly through its involvement in oxidative stress response and osteogenic differentiation.

[0123] However, the role of AMBP in the pathogenesis of CAVD has not been extensively investigated. To validate the effect of AMBP on osteoblastic differentiation of VICs, we performed functional analyses using primary VICs. Our results showed that downregulation of AMBP significantly increased osteoblastic differentiation under osteogenic conditions, confirming the protective effect of AMBP against calcium deposition and calcified nodule formation. In this study, we investigated the involvement of the MAPK pathway in mediating the protective effect of AMBP on osteoblastic differentiation. Previous studies have highlighted the significance of MAPKs in regulating osteoblastic differentiation. Our results showed that AMBP knockdown increased the phosphorylation levels of ERK1 / 2 and JNK, while the phosphorylation level of p38 remained unchanged. By using specific inhibitors targeting P-JNK and P-ERK1 / 2, we determined that the protective effect of AMBP on osteoblastic differentiation was mediated through these pathways. The protective effect of AMBP was further supported by in vivo experiments. Overexpression of Ambp in ApoE- / - mice significantly reduced aortic valve calcification and fibrosis and improved valvular function. These protective effects were attenuated by specific agonists of the P-ERK1 / 2 and p-JNK pathways, suggesting that AMBP protects AV function through these MAPK pathways. Interestingly, our results differ from previous reports on the P-p38 MAPK pathway in osteoblast differentiation, emphasizing the key roles of the P-ERK1 / 2 and p-JNK MAPK pathways. In this study, we explored how AMBP inhibits the phosphorylation of ERK1 / 2 and JNK in VIC cells.

[0124] The UPS (ubiquitin proteasome system) is responsible for the degradation of more than 80% of intracellular proteins. In addition to its important role in normal physiological processes, the UPS is also crucial in various pathological conditions. In this study, we found that the UPS mediated the inhibitory effect of AMBP on P-JNK and P-ERK1 / 2. Using PPI databases combined with the DEG set identified in this study, we identified FHL3 as a key protein that interacts with P-JNK and P-ERK1 / 2. In addition, we also found that FHL3 can bind to P-JNK and P-ERK1 / 2, thereby protecting them from UPS-mediated degradation. Co-IP experiments showed that AMBP competitively binds to FHL3, thereby promoting the ubiquitination and degradation of P-JNK and P-ERK1 / 2 in OM-activated VICs. We further identified the ZF domain as the key domain required for the interaction between FHL3 and AMBP. These findings highlight the potential therapeutic significance of AMBP as a target for the management of CAVD and provide new insights into the involvement of P-ERK1 / 2 and p-JNK pathways in mediating the protective effects of AMBP on osteoblast differentiation.

[0125] Notably, bortezomib, an ERK1 / 2 activator, partially reduced the protective effect of AMBP against CAVD. Given that bortezomib is a drug used to treat multiple myeloma, clinicians should be alert to the progression of aortic valve calcification in such patients. Administration of AMBP may mitigate this progression. This finding highlights a critical issue in the field of cardio-oncology that warrants further investigation of the interplay between cancer treatment and cardiovascular health.

[0126] This study has several limitations. First, the functional roles of the other six key hub genes identified in this study in osteoblast differentiation of VICs remain unexplored. Further studies are needed to elucidate the contribution of these genes to the pathogenesis of CAVD. Second, due to the lack of a VIC-specific promoter, we did not use VIC-specific overexpression AAV vectors or conditional knockout mice in this study. Future studies should focus on validating these findings using conditional AMBP knockout animal models to provide more precise insights into the role of the gene in CAVD. Finally, we did not measure the concentration of AMBP in the serum of patients, so its potential as a biomarker for the development, progression, or severity of CAVD is unclear. Future studies should evaluate serum AMBP levels in patients with CAVD to assess its potential as a clinical biomarker. These limitations highlight important directions for future research, which are essential to advance our understanding of CAVD and develop effective treatment strategies.

[0127] in conclusion

[0128] In conclusion, our study identified AMBP as a key regulator of CAVD pathogenesis. Through comprehensive RNA-seq analysis and experimental validation, we highlighted the important role of AMBP in regulating osteoblast differentiation and calcification in VICs via the MAPK signaling pathway, particularly by inhibiting ERK1 / 2 and JNK phosphorylation. In vivo overexpression of AMBP protected against aortic valve calcification in ApoE- / - mice, further validating its potential therapeutic relevance. Mechanistically, AMBP competitively bound to the ZF domain of FHL3, which abolished the inhibitory effect of FHL3 on the ubiquitin-proteasomal degradation of P-JNK and P-ERK1 / 2, thereby increasing the ubiquitin-proteasomal degradation of P-JNK and P-ERK1 / 2 in OM-activated VICs.

[0129] Despite the limitations in the exploration of other hub genes, the lack of a VIC-specific genetic model, and the unmeasured serum AMBP levels, our findings provide a strong basis for future studies. These results highlight the potential of AMBP not only as a therapeutic target but also as a biomarker for CAVD, paving the way for new intervention strategies. Continued research is essential to expand our understanding of the molecular mechanisms of CAVD and facilitate the translation of these findings into clinical applications.

[0130] Clinical perspective

[0131] This study identifies AMBP as a key regulator in calcific aortic valve disease (CAVD) and demonstrates its protective effects against valvular calcification and fibrosis by inhibiting ERK1 / 2 and JNK phosphorylation. These findings provide new insights into the molecular mechanisms of CAVD and suggest that AMBP may serve as a novel therapeutic target. Clinicians may consider the potential of AMBP-based therapies to protect calcium deposits in valvular interstitial cells (VICs), addressing a major unmet need in the management of CAVD. Furthermore, these results highlight the importance of incorporating molecular-level interventions into standard clinical practice for the management of CAVD, particularly in patients who do not respond to conventional lipid-lowering therapies.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of substances that promote overexpression of AMBP in the preparation of products for treating aortic valve calcification and / or fibrosis.

2. Application of substances that promote overexpression of AMBP in the preparation of products that inhibit the phosphorylation of ERK1 / 2 and JNK in VIC cells.

3. A pharmaceutical composition, characterized in that: It comprises the substance promoting overexpression of AMBP as claimed in claim 1 and / or at least one other non-drug active ingredient.

4. The pharmaceutical composition according to claim 3, characterized in that: The non-pharmaceutical active ingredient is a carrier, excipient or diluent commonly used in pharmacy.

5. The pharmaceutical composition according to claim 4, characterized in that: The carrier, excipient or diluent is selected from at least one of calcium silicate, lactose, sucrose, sorbitol, erythritol, cellulose, calcium phosphate, mannitol, starch, gum arabic, methylcellulose, gelatin, glucose, maltitol, talc, xylitol, polyvinyl pyrrolidone, microcrystalline cellulose, methyl hydroxybenzoate, alginate, propyl hydroxybenzoate, water, magnesium stearate or mineral oil.

6. The pharmaceutical composition according to claim 3, characterized in that: The dosage form of the pharmaceutical composition is an external preparation, an oral preparation, a suppository or an injection.

7. The pharmaceutical composition according to claim 6, characterized in that: The oral preparation is a tablet, powder, syrup, suspension, granule, emulsion, capsule or spray.

8. The pharmaceutical composition according to any one of claims 3 to 7, characterized in that: The pharmaceutical composition is used in preparing aortic valve calcification and / or fibrosis products.

9. The pharmaceutical composition according to claim 8, characterized in that: The product is a drug or a test reagent.

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