CN110433282B - Application of glucagon-like peptide-1 in preparation of medicine for treating calcified aortic valve diseases - Google Patents

Abstract

The invention belongs to the field of medicines, and relates to application of glucagon-like peptide-1 in preparation of medicines for treating calcified aortic valve diseases. The glucagon-like peptide-1 can inhibit and/or reverse calcification of valve interstitial cells and age-induced calcified aortic valve diseases by inhibiting differentiation and/or calcification of the valve interstitial cells to osteogenesis direction and/or inhibiting alkaline phosphatase activity and/or regulating and controlling expression of genes related to calcification of the valve interstitial cells, and can be used as a protective factor of calcified aortic valve diseases to prepare medicines for treating calcified aortic valve diseases, and the application prospect is wide.

Description

Application of glucagon-like peptide-1 in preparation of medicine for treating calcified aortic valve diseases

Technical Field

The invention belongs to the field of medicines, and relates to application of glucagon-like peptide-1 in preparation of medicines for treating calcified aortic valve diseases.

Background

Calcified Aortic Valve Disease (CAVD) is a common chronic heart valve disease characterized by degeneration and mineralization of the aortic valve, primarily characterized by thickening and calcification of the aortic valve. CAVD includes chronic inflammatory responses, lipid deposition, extracellular matrix remodeling, and activation of osteogenesis-related genes. Age-related degradation as an important risk factor for CAVD can lead to attenuation of some protective factors. CAVD is ubiquitous in the elderly, but there is currently no effective way to inhibit its progression.

Valve stromal cells (VICs) are a heterogeneous group of cells with multiple phenotypes (myofibroblasts, fibroblasts, and smooth muscle cells) that are involved in the physiological functioning of the aortic valve. For example: the risk factors of CAVD activate the osteogenic signaling pathway, further transforming valve stromal cells (qVICs) in the quiescent phase into activated valve stromal cells (aVICs ) and inducing their differentiation into osteogenic valve stromal cells (obVICs).

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted into plasma by enteroendocrine L-cells (terminal ileum and colon) and K-cells (duodenum and jejunum), and has pharmacological activity for regulating metabolic and cardiovascular diseases. GLP-1 is derived from glucagon and is a hormone consisting of 30 amino acids, which affects insulin sensitivity during blood glucose regulation, and this action may partially reverse aging-related degenerative diseases. GLP-1 has demonstrated its benefits for cardiovascular disease function, whether in preclinical experiments or clinical trials. However, the relationship between GLP-1 and CAVD has not yet been elucidated.

GLP-1 inhibits mineralization of valve smooth muscle cells by inhibiting arterial calcification by reducing expression of osteogenic genes. Phenotypic changes in VICs are the major events leading to calcified disease of the aortic valve, however it is not known whether GLP-1 regulates the phenotype of VICs.

Disclosure of Invention

The invention aims to provide a new application of glucagon-like peptide-1 (GLP-1).

Calcific aortic valve disease, CAVD, is prevalent in the elderly, but there is currently no method available to effectively inhibit its progression. GLP-1 is low expressed in plasma and tissues of CAVD patients. Clinical and cytological evidence suggests that GLP-1 is involved in the pathological process of aortic valve calcification. The invention discloses novel characteristics of GLP-1 and application thereof in preparing medicaments for treating CAVD.

The glucagon-like peptide-1 can be used for preparing medicaments for treating calcific aortic valve diseases.

Particularly, the glucagon-like peptide-1 is used as an active ingredient for inhibiting and/or reversing aortic valve interstitial cell calcification, an active ingredient for a calcified aortic valve disease protection factor, an active ingredient for inhibiting and/or reversing calcified aortic valve disease, and an active ingredient for inhibiting and/or reversing age-induced calcified aortic valve disease.

The glucagon-like peptide-1 can inhibit differentiation and/or calcification of valve interstitial cells to osteogenesis direction, and/or inhibit activity of alkaline phosphatase (ALP), and/or regulate and control gene expression related to calcification of valve interstitial cells, thereby inhibiting or reversing aortic valve interstitial cell calcification, inhibiting or reversing age-induced calcified aortic valve diseases, and can be used as a protective factor of calcified aortic valve diseases to prepare medicines for treating calcified aortic valve diseases.

Glucagon-like peptide-1 attenuates valvular stromal cell calcification and decreases alkaline phosphatase (ALP) activity, and glucagon-like peptide-1 exhibits a dose-dependent trend in attenuation of valvular stromal cell calcification and decrease in ALP activity, with a greater dose of glucagon-like peptide-1 showing a more pronounced attenuation of valvular stromal cell calcification and decrease in ALP activity. When the concentration reaches 25pmol/L or higher, the calcification of the aortic valve can be obviously inhibited, even the calcification of valve interstitial cells can be reversed, and the risk of calcified aortic valve diseases is reduced.

Glucagon-like peptide-1 significantly attenuated the tendency of valvular stromal cell calcification which increased over time and decreased the tendency of ALP activity which increased over time, and at a certain concentration (100 pmol/L in one embodiment of the invention) was effective in reversing age-induced valvular stromal cell calcification and decreasing the risk of age-induced calcified aortic valve disease.

The glucagon-like peptide-1 regulates and controls the expression of the genes related to the calcification of valve interstitial cells by inhibiting the expression of genes related to osteogenesis and/or improving the expression of genes related to osteogenesis resistance, inhibits the calcification of the valve interstitial cells and reduces the risk of calcified aortic valve diseases. The osteogenesis related genes are RUNX2, MSX2, BMP2 and BMP4, and the anti-osteogenesis related gene is SOX9.

The glucagon-like peptide-1 can inhibit calcification of valve interstitial cells, reduce the risk of calcified aortic valve diseases, especially age-related calcified aortic valve diseases, and has wide medical application prospect.

The present invention also provides a pharmaceutical preparation for preventing or treating calcified aortic valve diseases, comprising glucagon-like peptide-1 as an active ingredient, for preventing and/or treating calcified aortic valve diseases.

The pharmaceutical preparation also comprises any one or more auxiliary active ingredients, wherein the auxiliary active ingredients are at least one of anti-cardiovascular disease active ingredients, cardiovascular maintenance active ingredients, anti-atherosclerosis active ingredients, anti-thrombosis active ingredients, blood fat reducing active ingredients, blood sugar reducing active ingredients or blood pressure reducing active ingredients. The auxiliary active ingredients assist glucagon-like peptide-1 in the prevention and/or treatment of calcified aortic valve diseases.

The cardiovascular diseases include but are not limited to arrhythmia, myocardial infarction, heart failure, hypertension, atherosclerosis and the like.

The pharmaceutical preparation also comprises any one or more pharmaceutically acceptable auxiliary materials, wherein the pharmaceutically acceptable auxiliary materials comprise carriers, excipients, disintegrants, lubricants, adhesives, flavoring agents, absorbents, cosolvent, buffering agents, penetration enhancers, stabilizing agents and the like.

The pharmaceutical preparation can be prepared into any pharmaceutically acceptable dosage form with the functions of prevention and/or treatment according to the needs. The pharmaceutically acceptable dosage forms include liquid dosage forms and solid dosage forms, and are suitable for gastrointestinal administration, injection administration and the like. Liquid dosage forms include, but are not limited to, injections, suspensions, granules, and the like, and solid dosage forms include, but are not limited to, tablets, capsules, powders, granules, and the like.

The invention also discovers that the content of glucagon-like peptide-1 in aortic valve and plasma of patients with calcified aortic valve diseases is obviously reduced, and indicates that the reduction of the level of glucagon-like peptide-1 in the aortic valve causes calcification of valve interstitial cells (aVICs). Thus, reagents for detecting glucagon-like peptide-1 can be used as reagents for diagnosing or aiding in the diagnosis of calcified aortic valve disorders.

Meanwhile, the glucagon-like peptide-1 and the reagent for detecting the glucagon-like peptide-1 can be used for screening the medicine for treating the calcified aortic valve diseases.

Compared with the prior art, the invention has the beneficial effects that:

GLP-1 levels in calcified valve tissue are significantly lower than in normal aortic valve tissue, and a decrease in GLP-1 is associated with CAVD, and GLP-1 is involved in the mineralization process of AVICs by regulating specific calcification genes. In vitro experiments, GLP-1 antagonizes AVIC calcification in a dose-or time-dependent manner, or regulates specific AVIC mineralization genes by down-regulating RUNX1, MSX2, BMP4 expression and up-regulating SOX9 expression, so as to resist AVIC mineralization. A certain level of GLP-1 plasma concentration (100 pmol/L) can reverse valvular stromal cell calcification and reduce the risk of calcified aortic valve diseases, especially age-related calcified aortic valve diseases. Therefore, GLP-1 is expected to be a new target point for CAVD treatment.

Drawings

FIG. 1 is a histological calcification area staining pattern, a GLP-1 distribution area staining pattern (A) and a GLP-1 density distribution pattern (B) of the calcified vein group (CAVD, n = 11) and the non-calcified Control group (Control Valves, n = 12) of example 1.

FIG. 2 is a bar graph (A) showing alizarin red S staining results, a bar graph (B) showing alizarin red S staining quantitation results, and a bar graph (C) showing ALP activity quantitation results of the calcified valve Control group (Con, control) and the GLP-1 group (12.5, 25, 50, 100 pmol/L) in example 2.

FIG. 3 shows alizarin red S staining pattern (A), alizarin red S staining quantitative determination result (B) and ALP activity determination result (C) after 7, 14 and 21 days of culture in calcified valve Control group (Con, control) and GLP-1 group (100 pmol/L) of example 3.

FIG. 4 shows the expression of the calcification-associated genes (RUNX 2, MSX2, SOX9, BMP2, BMP 4) in the calcified-valve Control group (Con, control) and the GLP-1 group (100 pmol/L) in example 4: mrna relative expression profile, b fluorescence immunoassay profile.

FIG. 5 is a Western blot and relative protein expression profiles of calcification-associated genes (RUNX 2, MSX2, SOX9, BMP2, BMP 4) in the calcified-valve Control group (Con, control) and GLP-1 group (100 pmol/L) in example 4.

Detailed Description

Materials: calcified human aortic valves were taken from 11 patients undergoing valvular replacement and aortic valve leaflets were taken as normal aortic valve samples from 12 transplanted hearts from patients receiving heart grafts. The study protocol was approved by the ethical committee of the seiku hospital, shanghai university of transportation, and written informed consent was obtained from all patients.

Reagents and antibodies: recombinant GLP-1 peptide (human, code SCP 0153), 3' -diaminobenzidine liquid substrate system (code # D3939), alkaline phosphatase diethanolamine activity kit (code AP 0100), alizarin red S (code A5533), masson stain kit (code HT 15) were purchased from Sigma-Aldrich company (MO, USA).

The primary antibody was used to detect GLP-1 (accession number ab22625, abcam, MA, USA) in Immunohistochemistry (IHC) or immunoblot analysis, and the secondary antibody was horseradish peroxidase conjugated anti-rabbit antibody (accession number 7074, cell Signaling Technology, MA, USA) or Alexa Fluor 594 or Alexa Fluor 488 conjugated anti-rabbit antibody (accession number R37119 or accession number A27034, thermo Fisher Scientific, NY, USA), DMEM F12 medium.

GLP-1 concentrations were compared using immunohistochemical analysis for 11 calcified valve tissues and 12 normal valve tissues. The role and the characteristics of GLP-1 in AVICs (aortic valve interstitial cell) calcification and the expression regulation and control characteristics of calcification genes are described.

And (3) data analysis: data analysis was performed using SPSS software (version: 20). All results were tested as a two-sided test, with p <0.05 considered significant. Demographic and clinical characteristics were compared between CAVD and control groups, categorical variables were measured using Fisher's accuracy test, and differences between groups were calculated using two independent sample t-test or Wilcoxon rank sum test. For ALP and ALZ (alizarin red S), repeated measures analysis of variance (ANOVA) was used as a fixed factor to compare the differences between the two groups.

Example 1 distribution of GLP-1 in calcified aortic valves

Immunohistochemical analysis methods were as follows:

human calcified aortic valves (CAVD, n = 11) and non-calcified aortic valves (control valves, i.e. normal, non-calcified, n = 12) were used for histological and immunohistochemical analysis. Serial frozen sections (5 μm thick) were made after treatment of the samples with 4% paraformaldehyde overnight. Sections were stained with hematoxylin and eosin (H & E), alizarin Red S (Alzarin Red S), and Masson (Masson) trichrome. The results are shown in fig. 1A, HE staining shows normal leaflets are thin, distinct in layer, no vessel in the valve, and continuous in fibrous layer; the CAVD valve blades thicken collagen fiber hyperplasia, the arrangement is disordered, elastic fibers are broken, the collagen fiber hyperplasia and the arrangement are disordered, some are arranged in a swirl shape, and the center of the fiber hyperplasia has glass-like degeneration. ARS staining shows that (red is positive staining) normal valve tissue is uniform blue and has no positive staining result, CAVD valve tissue has obvious red staining on the whole, and positive staining on calcified parts is more obvious. Masson staining showed that normal valves were arranged in three layers with no apparent collagen fiber proliferation (collagen fibers are blue); the CAVD lesion staining valve has the defects that collagen hyperplasia and arrangement disorder are seen in the non-calcified part, the collagen hyperplasia of the calcified part is serious, the arrangement is more disordered, and inflammatory cell infiltration is seen under endothelium. GLP-1 immunohistochemical staining shows that GLP-1 is uniformly and strongly expressed in normal valves and is obviously reduced in CAVD valve tissues. According to histological analysis, calcified aortic valves show structural thickening, mineralization and reconstruction of the extracellular matrix.

GLP-1 in the valve was detected by immunohistochemistry using the anti-GLP-1 antibody, as shown in FIG. 1A. After incubation with horseradish peroxidase (HRP) conjugated secondary antibody (1. Immunohistochemical analysis showed that GLP-1 was distributed mainly in the enriched and unmineralised areas of the VICs, whether in the non-calcified Control group (Control) or the calcified valve group (CAVD).

The integrated optical density levels of GLP-1 were calculated for the 12 non-calcified Control (Control Valves) and 11 calcified valve (CAVD) groups, and the results are shown in FIG. 1B. Non-calcified control group: 9170 ± 695.9, calcified valve group: 5606 ± 750.4, p =0.0042. The GLP-1 concentration of the calcified valve group was reduced by 39% compared to the non-calcified control group. Thus, there is a relationship between GLP-1 and CAVD.

Example 2

Based on the results of the correlation of GLP-1 to CAVD, we treated AVICs with different doses of GLP-1 to identify the effect of GLP-1 on calcification in vitro experiments.

2.1 Primary culture of valvular stromal cells

Human aortic valve leaflets (normal) from heart grafts were taken for primary culture of AVICs. Briefly, the leaflets are digested with collagenase and gently scraped to expose the endothelial layer. Then cut into small pieces of 1-2 square centimeters, add 20% fetal bovine serum, L-glutamine (2 mmol/L), penicillin (100U/mL) and streptomycin (100. Mu.g/mL) in DMEM: F12 (1 ₂ And cultured at 37 ℃. When adherent growth reached 80% confluency, AVIC was passaged using trypsin-EDTA. AVICs from passage 3 to 8 were used for the experiments.

2.2. In vitro testing for calcification of AVICs

Primary AVICs were isolated from normal group human aortic valves and 3 to 8 passage AVICs were used for experiments. AVIC calcification was induced in osteogenic medium containing DMEM supplemented with 15% FBS, 50mg/mL ascorbic acid-2-phosphate, 10nM dexamethasone and 10mM beta-glycerophosphate, the medium being changed every 48-72 hours.

GLP-1 content in the culture medium was 0 (calcified valve control group, con group), 12.5pmol/L, 25pmol/L, 50pmol/L, and 100pmol/L, respectively.

Cells cultured for 3 weeks were collected. Calcification of AVIC was detected by alizarin red S staining: the cells were washed with distilled water and then placed in fresh 2% alizarin red S (pH 4.1-4.3) for 5 minutes (positive staining results for red/orange) as shown in FIG. 2A: con group had a clear red stain, GLP-1 group decreased the number of ARS positive staining cells with increasing concentration. Alizarin S staining indicated that osteogenic media induced AVIC mineralization, but higher doses (25-100 pmol/L) of GLP-1 could reverse AVIC calcification.

2.3 detection of AVIC calcification

To quantify alizarin red S staining, the dye was released from the cytoplasmic matrix by incubation with cetylpyridinium chloride for 15 minutes, and the released dye was quantified spectrophotometrically at 540 nm.

The activity of alkaline phosphatase ALP is an important marker for osteoblast differentiation, and the activity of alkaline phosphatase (ALP) was determined by measuring the level of p-nitrophenol in AVIC using a spectrophotometric method, and the amount of alizarin red S staining and ALP activity was normalized according to the total amount of cellular proteins. The results are shown in FIGS. 2B and 2C.

The staining results of alizarin S dilutions are shown in fig. 2B: calcified valve Control group (Con, control) =213.5 ± 9.248 μ g/mg; GLP-1 (12.5 pmol/L) =203.7 ± 7.535 μ g/mg, relative calcified valve control group P =0.318; GLP-1 (25 pmol/L) =176.3 ± 5.754 μ g/mg, relative calcified valve control group P =0.033; GLP-1 (50 pmol/L) =149.7 ± 7.632 μ g/mg, relative calcified valve control group P =0.007, GLP-1 (100 pmol/L) =101.7 ± 8.950 μ g/mg, relative calcified valve control group P =0.001.

The ALP activity assay results are shown in FIG. 2C: calcified valve Control group (Con, control) =605.5 ± 20.53U/mg; GLP-1 (12.5 pmol/L) =595.7 ± 18.26U/mg, relative calcified valve control P =0.513; GLP-1 (25 pmol/L) =519.5 ± 12.73U/mg, relative calcified valve control group P =0.002; GLP-1 (50 pmol/L) =358.7 ± 17.94U/mg, relative calcified valve control P =0.001; GLP-1 (100 pmol/L) =218.3 + -12.41U/mg, relative calcified valve control group P <0.001.

The results all indicate that GLP-1 reduces AVIC calcification in a dose-dependent manner.

Example 3GLP-1 is associated with age-induced calcification

In vitro experiments, AVICs calcification was induced at different times of 7-21 days to mimic the age-induced calcification process.

AVICs were performed in parallel with example 2 in GLP-1 group (100 pmol/L) supplemented with GLP-1 and calcified valve Control (Con, control) group (Con, control) not supplemented with GLP-1. The method for detecting AVIC calcification was the same as in example 2.

Alizarin S staining results are shown in fig. 3A, with more pronounced red-brown positive staining of cellular calcification, indicating that the progression of calcification increases in rate and severity over time.

Results of alizarin S staining quantitative analysis time effect are shown in fig. 3B, calcified valve Control group (Con, control): 7 days =129.0 ± 9.2 μ g/mg,14 days =161.0 ± 6.4 μ g/mg,21 days =218.5 ± 7.6 μ g/mg; GLP-1 (100 pmol/L) group: 7 days =79.0 ± 7.1 μ g/mg,14 days =91.3 ± 10.8 μ g/mg,21 days =108.3 ± 10.5 μ g/mg, respectively, compared to a parallel arrangement of a GLP-1-unsupplemented calcified valve control group: p <0.01; the addition of GLP-1 significantly attenuated the tendency to calcify over time compared to the calcified valve control group.

ALP activity assay results are shown in fig. 3C, calcified valve Control (Con, control): 7 days =350.8 ± 28.2U/mg,14 days =495.8 ± 37.2U/mg,21 days =624.8 ± 40.9U/mg; GLP-1 (100 pmol/L) group: 7 days =147.2 ± 21.3U/mg,14 days =174.3 ± 19.1U/mg,21 days =212.8 ± 26.5U/mg, compared to a control group of calcified valves not treated with GLP-1 arranged in parallel: p <0.01. The addition of GLP-1 significantly attenuated the tendency to calcify over time compared to the calcified valve control group.

Example 4GLP-1 regulates the expression of calcification-related genes

Many genes are involved in the mineralization process of AVIC, RUNX2, MSX2 and SOX9 play a role as nuclear transcription factors in downstream gene transcription. For example, the target genes BMP2 and BMP4 may promote calcification of AVICs. Therefore, we examined whether GLP-1 could inhibit the calcification of AVIC by controlling the expression of these genes, and placed a calcified valve Control group (Con, control) and a GLP-1 group cultured by adding GLP-1 (100 pmol/L) in parallel.

1. Quantitative real-time PCR analysis

Levels of RUNX2, MSX2, SOX9, BMP2 and BMP4 mrnas were detected by real-time PCR.

Total RNA was extracted, 5. Mu.g of total RNA was reverse transcribed into cDNA using a reverse transcription system ((Promega Corp., WI, USA). PCR amplification was performed in the StepOne system (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems, calif., USA). Gene expression levels were normalized to β -actin and data were analyzed using StepOne software v2.1 (Applied Biosystems). Results are shown in FIG. 4A.

The results show that GLP-1 decreased transcription of RUNX2 by 62% (calcified valve control group =1.01 ± 0.02VS GLP-1=0.38 ± 0.04, p-restricted 0.01), MSX2 transcription by 54% (calcified valve control group =1.00 ± 0.02 VS-1 =0.46 ± 0.06, p-restricted 0.01), BMP2 transcription by 46% (calcified valve control group =1.00 ± 0.01VS GLP-1=0.54 ± 0.06, p-restricted 0.01) and BMP4 transcription by 59% (calcified valve control group =1.01 ± 0.03VS GLP-1=0.41 ± 0.02, p-restricted 0.01), but increased transcription of SOX9 by 2.01 times (calcified valve control group =1.00 ± 0.02 GLP-1=2.01 ± 0.14, p-restricted 0.01).

2. Immunohistochemical analysis

The membranes were blocked with anti-RUNX 2 (1. Then incubated with horseradish peroxidase-labeled secondary antibody (1.

Distribution of RUNX2, MSX2, SOX9, BMP2 and BMP4 was examined by fluorescence immunoassay, and as shown in fig. 4B, RUNX2, MSX2 and SOX9 localized in the nucleus, and BMP2 and BMP4 were expressed in each structure of the cell. GLP-1 decreases the expression levels of RUNX2, MSX2, BMP2, and BMP4 but increases the expression level of SOX9.

3.Western blot

The concentration of the above proteins was detected by western blot and analyzed by IOD value (ratio of target protein/β -actin).

The cells were lysed with a ProteJET mammalian cell lysis reagent (Fermentas, md., USA) to extract cytoplasmic proteins. Equal amounts of protein extracts were treated with 10% SDS/PAGE and blotted onto polyvinylidene fluoride membranes. The membranes were blocked with anti-RUNX 2 (1. Then incubated with horseradish peroxidase-labeled secondary antibody (1. Blots were developed using an ECL detection system (Millipore, MA, USA). Each image was taken and the intensity of each band was analyzed using QuantityOne (Bio-Rad) software. The results are shown in FIG. 5.

The concentrations of these proteins were measured by western blot and analyzed by IOD value (ratio of target protein/β -actin), and the results are shown in fig. 5. GLP-1 decreased expression of RUNX2 by 49% (calcified valve control group =2.21 ± 0.09VS GLP-1=1.12 ± 0.17, p-herding 0.01), expression of MSX2 by 53% (calcified valve control group =1.75 ± 0.08VS GLP-1=0.83 ± 0.07, p-herding 0.01), expression of BMP2 by 57% (calcified valve control group =1.38 ± 0.13VS GLP-1=0.60 ± 0.10, p-herding 0.01), expression of BMP4 by 48% (calcified valve control group =1.70 ± 0.09 GLP-1=0.89 ± 0.16, p-herding 0.01), but expression of SOX9 increased by 1.98 times (calcified valve control group =0.90 ± 0.13 GLP-1=1.78 ± 0.13, p-herding 0.01).

Conclusion

Although CAVD is well understood by some clinical, genetic and animal studies, more important advances (e.g., optimal diagnostic and therapeutic strategies) have not been completed. Still, a more profound academic exploration of the field is needed, especially in the direction of the important endogenous protection factors. In this study, we found that GLP-1 was reduced in concentration in the aortic valve of CAVD patients. GLP-1 also reduces the mineralization of AVIC by modulating calcification-related genes. Thus, GLP-1 has a protective effect against CAVD.

1. GLP-1 can be used as a novel CAVD-related protective factor

GLP-1 is an incretin hormone secreted into the plasma by enteroendocrine L-cells (terminal ileum and colon) and K-cells (duodenum and jejunum); however, we found the presence of GLP-1 in the area of the unmineralised aortic valve with or without calcific foci, suggesting that GLP-1 is secreted by intestinal cells and then recruited to the aortic valve, thus affecting the function of AVICs. GLP-1 is located in interstitial spaces and tissues to regulate metabolic diseases such as diabetes and obesity, while GLP-1 also regulates cellular function to protect the body from cardiovascular disease. In vitro and in vivo experiments, studies of atherosclerosis have shown that GLP-1 promotes vasodilation, inhibits inflammatory responses in endothelial cells, suppresses lipid uptake and inflammatory activity of macrophages, and inhibits proliferation of Smooth Muscle Cells (SMCs) to prevent progression of atherosclerosis. In arterial calcification, similar to the process of bone formation, the event of differentiation of Vascular Smooth Muscle Cells (VSMCs) into osteoblastic phenotype plays a key role in arterial calcification, while GLP-1 can inhibit osteoblastic differentiation and calcification of human VSMCs. Although CAVD has some similarities with arterial calcification, differentiation and mineralization of aortic valve interstitial cells are the main cytopathological processes of CAVD, unlike the process of arterial calcification.

In this study, we found that GLP-1 was reduced by 39% in calcified valves (FIG. 1), indicating that reduced GLP-1 levels in the aortic valve lead to calcification of AVIC. However, it is not known whether GLP-1 can reverse CAVD by modulating AVIC differentiation and calcification into osteogenic directions. To explore this problem, we added different doses of GLP-1 to AVICs during the normalization process of calcification. High doses of GLP-1 significantly reduced alizarin S densitometry and ALP activation (fig. 2), demonstrating that GLP-1 can mitigate CAVD by preventing mineralization of AVICs. Our findings indicate that GLP-1 inhibits calcification of AVICs and shows a protective effect in CAVD.

2. GLP-1 can reverse age-induced calcification in CAVD

CAVD is a chronic degenerative disease with a variety of risk factors including diabetes, hypertension, dyslipidemia, and nephropathy. Multiple ethnic studies (MESA studies) of atherosclerosis found the highest incidence of CAVD in whites other than hispanic, followed by hispanic and black, suggesting that CAVD exhibited ethnic differences. Our research in the Chinese population found that: age, fasting plasma glucose, hbA1c, HDL, BUN, and GLP-1 are independent risk factors for CAVD, and also suggest that age, diabetes, dyslipidemia, and renal insufficiency are associated with CAVD in the Chinese population.

Although there are many pathological factors involved in the onset of CAVD, age is an important irreversible risk factor and has strong correlation with CAVD. Previous studies have shown that more than 50% of patients with aortic valve calcification are older than 75 years, while 2-3% of this elderly population develop severe valvular stenosis. As the time of in vitro calcification culture increased, mineralization levels of AVICs increased and GLP-1 decreased time-dependent AVICs calcification (FIG. 3). These results all demonstrate that age plays a key role in CAVD mineralization, and GLP-1 also reduces age-induced AVICs calcification.

3. GLP-1 regulatable AVIC mineralization related gene

RUNX2, MSX2, SOX9, BMP2 and BMP4 are important proteins associated with calcification. RUNX2 is a transcription factor for bone and cartilage formation and is regulated in a variety of ways. RUNX2 is up-regulated during atherosclerotic calcification and endochondral mineralization. Hydrogen peroxide can activate osteogenic Cbfa1/RUNX2 and MSX2/Wnt pathways, thereby promoting the mineralization process. Miller et al also found that these two regulatory cascades are activated upon calcification of the human aortic valve. Garg et al demonstrated that Notch1 inhibits osteogenic mineralization in AVICs by maintaining expression of SOC 9. BMP2 and BMP4 increase OPN secretion by upregulating ALP leading to degradation of pyrophosphate in the tissue. GLP-1 inhibits osteoblast differentiation by inhibiting ALP, osteocalcin and RUNX2 in human VSMCs. However, it is not clear whether GLP-1 modulates the expression of MSX2, SOX9, BMP2 and BMP 4.

We found in this experiment that GLP-1 decreased the expression of RUNX2, MSX2, BMP2 and BMP4 but increased the expression of SOX9 in activated valvular stromal cells AVICs (FIG. 4), this result revealing for the first time the association of GLP-1 and these genes during AVIC calcification. The above results indicate that GLP-1 can counteract the mineralization of AVICs via two pathways, the first pathway being accomplished by inhibiting the expression of osteogenesis-related genes, the other pathway being accomplished by promoting the expression of anti-osteogenic genes.

4. Application prospect

The present study shows that GLP-1 levels are reduced both in calcified aortic valves and in plasma of CAVD patients, and this reduction in concentration is age-related. This indicates that GLP-1 is valuable for predicting CAVD onset and development.

Valve replacement is currently the effective treatment for CAVD, however, this treatment also results in high surgical risk and partial postoperative recurrence for the patient. Previous randomized trials have shown that statins have no therapeutic effect on aortic stenosis. No therapies that can inhibit the progression of CAVD have been found, particularly against age-induced valve degeneration, and remain unresponsive. However, our experimental results demonstrate that high levels of GLP-1 plasma concentrations can reduce the risk of cav, especially for age-related cav.

In vitro experiments on AVICs also indicate that high doses of GLP-1 inhibit AVIC calcification. GLP-1 was found to protect against cardiovascular disease and to improve postoperative recovery in vivo. Continuous GLP-1 infusion (1.5 pmol/kg/min) -12 hours before to 48 hours after surgery gives better control of blood glucose in patients undergoing coronary artery bypass surgery, and in addition to reducing the onset of arrhythmia, also reduces the infusion of inotropic or vasoactive drugs to promote post-operative hemodynamic recovery. In patients with acute myocardial infarction and left ventricular ejection fraction <40%, the addition of GLP-1 (1.5 pmol/kg/min) to the basal treatment drug increased the left ventricular ejection fraction, global wall motion score index and regional wall motion score index after successful angioplasty compared to the control group. Furthermore, GLP-1 plus basal drug infusion (2.5 pmol/kg/min) at 5 weeks in NYHA grade III/IV heart function heart failure patients increased left ventricular ejection fraction in both diabetic and non-diabetic patients. These studies are expected to provide further insight into the role that GLP-1 therapy plays in the prevention of CAVD.

Claims (7)

1. Application of glucagon-like peptide-1 in preparing medicine for treating calcified aortic valve diseases is provided.

2. The pharmaceutical use of glucagon-like peptide-1 according to claim 1, wherein said glucagon-like peptide-1 is used as an active ingredient of a protective factor for calcified aortic valve diseases, an active ingredient for inhibiting and/or reversing age-induced calcified aortic valve diseases, an active ingredient for inhibiting and/or reversing calcification of interstitial cells of aortic valve.

3. The pharmaceutical use of glucagon-like peptide-1 according to claim 1 or 2, wherein said glucagon-like peptide-1 inhibits differentiation and/or calcification of valvular stromal cells into osteogenic direction, and/or inhibits the activity of alkaline phosphatase, and/or modulates valvular stromal cell calcification-related gene expression.

4. The pharmaceutical use of glucagon-like peptide-1 according to claim 3, wherein said glucagon-like peptide-1 regulates the expression of a gene associated with calcification of valve stromal cells as an active ingredient for inhibiting the expression of a gene associated with osteogenesis and/or promoting the expression of a gene associated with anti-osteogenesis.

5. The pharmaceutical use of glucagon-like peptide-1 according to claim 4, wherein said osteogenesis related gene is RUNX2, MSX2, BMP2 and BMP4 and said anti-osteogenesis related gene is SOX9.

6. The glucagon-like peptide-1 or the detection reagent of the glucagon-like peptide-1 is used for screening the medicine for treating calcified aortic valve diseases.

7. The detection reagent of the glucagon-like peptide-1 is used for preparing a reagent for diagnosing or assisting in diagnosing calcified aortic valve diseases.

Images