Application of NFATc3 as aortic dissection and aneurysm drug target

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a medicine for inhibiting eEF2 in aortic dissection and aneurysm medicine and application of NFATc3 serving as an aortic dissection and aneurysm medicine target. The invention provides an application of NFATc3 as an aortic dissection and aneurysm drug target. The relationship between the NFATc3 and the aortic dissection and the aneurysm is found for the first time, specifically, the expression of the NFATc3 in the VSMCs of the human aortic aneurysm is obviously up-regulated, and the expression of the NFATc3 of cytoplasm and cell nucleus is obviously increased in human and mouse AAD tissues. Based on the discovery, the invention provides a new application of a targeted NFATc3 drug in preparation of drugs for preventing or / and treating aortic dissection and aneurysm. Experiments show that the development of aortic dissection and aneurysm can be significantly slowed down by inhibiting the expression or function of NFATc3.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of NFATc3 as a drug target for aortic dissection and aneurysm. Background Art

[0002] Aortic aneurysm and dissection (AAD) are fatal diseases with extremely high mortality rates. According to literature statistics, within the first 1-3 days after the onset of AAD, the mortality rate increases by 1-2% every hour. For AAD patients without active treatment, the mortality rate within 48 hours after the onset can be as high as 50%. In addition, thoracic aortic dissection (TAD) and abdominal aortic aneurysm (AAA) are the most common clinically. TAD and AAA exhibit many similar pathological changes, including degradation of the extracellular matrix (ECM), phenotypic transformation of vascular smooth muscle cells (VSMCs), reduction in the number of VSMCs, and chronic inflammatory responses.

[0003] Under the action of MMPs, a large amount of elastic fibers and collagen fibers in the aortic media layer are degraded, resulting in changes in the composition and proportion of extracellular matrix components, thereby inducing AAD.

[0004] The phenotypic transformation of VSMCs from the contractile type to the synthetic type will lead to the degradation and degeneration of the aortic structure. The combined action of EMC degradation and VSMC phenotypic transformation can cause damage and loss of function of the aortic media layer that maintains vascular wall tension, ultimately leading to the occurrence of AAD.

[0005] NFATc3 is nuclear factor of activated T cells 3, a key transcription factor in the NFAT family, and plays a key role in calcium-dependent gene regulation. Although initially studied for its role in T cell activation and immune responses, NFATc3 has since played a key role in cardiovascular physiology, particularly in heart diseases. Its activation is mediated by the calcium signaling pathway, which triggers its dephosphorylation and translocation to the nucleus, where it regulates the expression of genes related to cell growth, survival, and differentiation.

[0006] Currently, there is no relevant literature publicly documenting the relationship between NFATc3 and aortic dissection and aneurysm. Summary of the Invention

[0007] (I) Technical Problems to be Solved

[0008] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides an application of a drug that inhibits eEF2 in drugs for aortic dissection and aneurysm, which provides a new application of a drug that inhibits eEF2;

[0009] Correspondingly, the present invention provides an application of NFATc3 as a drug target for aortic dissection and aneurysm, which provides a new application of a drug targeting NFATc3 and can be used to prepare drugs for preventing and treating aortic dissection and aneurysm;

[0010] Correspondingly, the present invention also provides NFATc3 expression level as a biomarker for AAD diagnosis or prognosis assessment;

[0011] Correspondingly, the present invention also provides a gene therapy vector.

[0012] (2) Technical solutions

[0013] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0014] In the first aspect, the present invention provides an application of a drug that inhibits eEF2 in drugs for aortic dissection and aneurysm,

[0015] The present invention discovers an increase in eEF2 and a decrease in the level of p-eEF2 in the aortic tissue of AAD, and discovers a new function of NFATc3 in regulating protein synthesis, which interacts with eEF2 at Thr56 and inhibits its phosphorylation in ribosomes without affecting the expression of eEF2K or PP2A. In addition, activating eEF2 with A-484954 reversed the slowdown of protein synthesis rate and VSMC phenotypic conversion caused by NFATc3 deficiency. More importantly, the upregulation of eEF2 reversed the protective effect in the progression of AAD caused by NFATc3 deficiency. On the contrary, inhibitors of eEF2, including cabamiquine (an inhibitor of eEF2), reversed the increase in protein synthesis and VSMC phenotypic conversion caused by NFATc3 overexpression. NFATc3 binds to eEF2 through RHR, that is, the DNA binding domain, reducing the phosphorylation level of eEF2, thereby increasing its activity to promote protein synthesis. Based on the above findings, the present invention provides a new application of a drug that inhibits eEF2 in the preparation of drugs for preventing and / or treating aortic dissection and aneurysm.

[0016] Optionally, the drug that inhibits eEF2 includes cabamiquine.

[0017] Carbamiquine, an antimalarial drug, targets eEF2 and inhibits protein synthesis to combat malaria parasites. Due to its effectiveness and safety, it is currently in a Phase I clinical trial for the treatment of Plasmodium falciparum sporozoites (https: / / clinicaltrials.gov; NCT04250363, NCT03261401)

[90] . In this study, the addition of carbamiquine significantly slowed down the phenotypic transformation of VSMCs in a NFATc3-dependent manner and inhibited the progression of the AAD model induced by BAPN, BAPN / AngII, and PCSK9DY / AngII. The results of in vivo animal experiments in this invention provide evidence for carbamiquine to pharmacologically inhibit eEF2 in the prevention and treatment of AAD.

[0018] In a second aspect, the present invention provides the use of NFATc3 as a drug target for aortic dissection and aneurysm.

[0019] The present invention discovers for the first time the relationship between NFATc3 and aortic dissection and aneurysm. Specifically, it is found that the expression of NFATc3 is significantly upregulated in VSMCs of human aortic aneurysms, and the cytoplasmic and nuclear expression of NFATc3 is significantly increased in human and mouse AAD tissues. Based on this discovery, the present invention provides a new application of drugs targeting NFATc3 in the preparation of drugs for preventing or / and treating aortic dissection and aneurysm.

[0020] Optionally, the drug is an inhibitor of NFATc3.

[0021] Among them, the inhibitors of NFATc3 include but are not limited to: VIVIT peptide, tacrolimus, cyclosporine A,

[0022] Among them, the VIVIT peptide is a cell-permeable polypeptide inhibitor that can selectively inhibit the dephosphorylation of NFAT mediated by calcineurin, thereby blocking the activation of NFATc3.

[0023] Optionally, the inhibitor acts by inhibiting the expression or / and function of NFATc3 in vascular smooth muscle cells.

[0024] Through experiments, the present invention discovers that by inhibiting the expression or / and function of NFATc3, the progression of aortic dissection and aneurysm can be significantly slowed down; and it can reduce the degradation of ECM in AAD and reduce the transformation of VSMCs from the contractile type to the synthetic type. Therefore, as long as a drug can inhibit the expression or / and function of NFATc3, it can be applied to the drugs for preventing, treating, or / and preparing aortic dissection and aneurysm.

[0025] Optionally, the drug is a compound that blocks the interaction between NFATc3 and eEF2.

[0026] Optionally, the drug specifically binds to the phosphorylation site of eEF2.

[0027] Optionally, the phosphorylation site of eEF2 is Thr56.

[0028] Optionally, the drug is a compound that regulates the transcription of matrix metalloproteinases MMP9 and MMP2.

[0029] The present invention first reveals the dual mechanism of action of NFATc3 in the pathology of AAD:

[0030] NFATc3 promotes ECM degradation by directly regulating the transcription of MMP9 / MMP2, inducing AAD;

[0031] NFATc3 binds to the eEF2 phosphorylation site through the RHR domain, reduces the phosphorylation level of eEF2, promotes ribosomal protein synthesis, induces the transformation of VSMCs into the synthetic type, and exacerbates the progression of AAD.

[0032] Therefore, the present invention further defines the drug as a compound that blocks the interaction between NFATc3 and eEF2. More specifically, it can be a compound targeting eEF2, including but not limited to carbamimidoyl quinoline or / and its pharmaceutical derivatives, eEF2 kinase inhibitors or / and their pharmaceutical derivatives.

[0033] Optionally, the drug is a compound that reduces the phosphorylation level of eEF2.

[0034] Optionally, the drug includes a gene editing tool or / and an RNA interfering agent targeting NFATC3.

[0035] In a third aspect, the present invention also provides an application of the NFATc3 expression level as a biomarker for the diagnosis or prognosis evaluation of AAD.

[0036] Specifically, by detecting the expression level of NFATc3 in the VSMCs or blood samples of a subject, when the expression level is higher than the healthy control threshold, it is determined as a high-risk or advanced stage of AAD.

[0037] Specifically, the detection method of the NFATc3 expression level includes immunohistochemical detection using a specific antibody against NFATc3, or quantitative PCR to detect its mRNA expression level.

[0038] In a fourth aspect, the present invention also provides a method for screening compounds against aortic dissection and aneurysm, which includes the following steps:

[0039] S1 Bind NFATc3 and the eEF2 phosphorylation site in vitro;

[0040] S2 Determine the inhibition rate of the compound to be screened on the binding;

[0041] S3 Screen out the compounds with an inhibition rate greater than 50% for animal model verification.

[0042] Among them, the method for the in vitro binding of the phosphorylation sites of NFATc3 and eEF2 includes, but is not limited to, detecting the binding activity by using fluorescence resonance energy transfer (FRET) technology.

[0043] In the fifth aspect, the present invention provides a gene therapy vector, which includes: an shRNA sequence targeting NFATc3 and a VSMC targeting delivery element. The targeting delivery element is preferably an AAV9 viral vector.

[0044] (III) Beneficial effects

[0045] The beneficial effects of the present invention are:

[0046] The present invention provides the application of the drug that inhibits eEF2 in the drugs for aortic dissection and aneurysm, and it can effectively treat aortic dissection and aneurysm.

[0047] The present invention provides a new application of the drug targeting NFATc3. The drug targeting NFATc3 can be used to prepare drugs for preventing and treating aortic dissection and aneurysm. Experiments show that inhibiting the expression or function of NFATc3 can significantly slow down the progression of aortic dissection and aneurysm.

[0048] The present invention also provides the application of the NFATc3 expression level as a biomarker for AAD diagnosis or prognosis evaluation. Description of the drawings

[0049] Figure 1 It is a schematic diagram for the construction of a mouse with specific knockout of NFATc3 in vascular smooth muscle cells in Example 7 of the present invention;

[0050] Figure 2 It is a graph of the experimental results that VSMC-NFATc3 knockout can reduce the occurrence of AAD in mice in Example 8 of the present invention;

[0051] Figure 3 It is a graph of the experimental results that VSMC-NFATc3 knockout can reduce the occurrence of AAA in mice in Example 8 of the present invention;

[0052] Figure 4 It is a graph of the experimental results that VSMC-NFATc3 knockout can restore the vasoconstriction and vasodilation functions reduced by BAPN modeling in Example 9 of the present invention;

[0053] Figure 5This is the experimental result graph showing that the deletion of VSMC-NFATc3 in Example 9 of the present invention reduces the phenotypic transformation of VSMCs from the contractile type to the synthetic type;

[0054] Figure 6 This is the experimental result graph of the in-vivo experiment in Example 11 of the present invention demonstrating that NFATc3 can up-regulate the expression of MMP2 and MMP9;

[0055] Figure 7 This is the result graph showing that NFATc3 can up-regulate the transcriptional levels of MMP9 and MMP2;

[0056] Figure 8 This is the result graph of the potential binding sites of NFATc3 in the MMP2 and MMP9 promoters in Example 11 of the present invention;

[0057] Figure 9 This is the experimental result graph of the experiment in Example 11 of the present invention that NFATc3 up-regulates the luciferase activities driven by the MMP9 and MMP2 promoters, and the mutation of the NFATc3 binding site down-regulates the NFATc3-dependent effect;

[0058] Figure 10 This is the experimental result graph showing that NFATc3 in VSMCs enriches H3K27ac and H3K4me3 located proximal to the MMP9 and MMP2 promoters;

[0059] Figure 11 This is the experimental result graph of the experiment in Example 12 of the present invention that NFATc3 can promote the phenotypic transformation of vascular smooth muscle cells from the contractile type to the synthetic type;

[0060] Figure 12 This is the experimental result graph of the experiment in Example 13 of the present invention that NFATc3 can bind to non-phosphorylated eEF2;

[0061] Figure 13 This is the experimental result graph of the experiment in Example 13 of the present invention that the knockout of VSMC-NFATc3 can increase the protein level of p-eEF2;

[0062] Figure 14 This is the experimental result graph of the experiment in Example 13 of the present invention that NFATc3 preferentially binds to non-phosphorylated eEF2;

[0063] Figure 15 This is the experimental result graph of the experiment in Example 13 of the present invention that NFATc3 can bind to eEF2 through its RHR domain;

[0064] Figure 16 This is the experimental result graph showing that NFATc3 increases the protein synthesis rate by inhibiting the phosphorylation of eEF2;

[0065] Figure 17This is the experimental result graph showing that carbamylquine can reduce the occurrence of TAAD in Example 14 of the present invention;

[0066] Figure 18 This is the experimental result graph showing that carbamylquine can reduce the occurrence of AAD in Example 14 of the present invention;

[0067] Figure 19 This is the experimental result graph showing that carbamylquine can reduce the occurrence of AAA in Example 14 of the present invention;

[0068] Figure 20 In Example 14 of the present invention, in NFATc3 smcKI This is the experimental result graph showing that carbamylquine can increase the expression of p-eEF2 in mice. Detailed implementation manners

[0069] To illustrate in detail the possible application scenarios, technical principles, specific implementable solutions, achievable objectives and effects of the present application, the following will be described in detail with reference to the specific examples listed. The examples described herein are only used to more clearly illustrate the technical solutions of the present application, so they are only examples and cannot be used to limit the protection scope of the present application.

[0070] Referring to "example" herein means that the specific features, structures or characteristics described in connection with the example may be included in at least one example of the present application. The term "example" appearing at various positions in the specification does not necessarily refer to the same example, nor does it particularly limit its independence or relevance to other examples. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each example can be combined in any way to form the corresponding implementable technical solution.

[0071] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the technical field to which the present application belongs; the use of the relevant terms herein is only for describing specific examples and is not intended to limit the present application.

[0072] Example 1

[0073] This example provides a drug for treating aortic dissection and aneurysm, which is an inhibitor of NFATc3, and the inhibitor is not limited to the combination of one or more of the following drugs: VIVIT peptide, tacrolimus, cyclosporin A, carbamylquine or / and its pharmaceutically derivatives, eEF2 kinase inhibitor or / and its pharmaceutically derivatives, gene editing tool, RNA interfering agent targeting NFATC3.

[0074] In some other specific embodiments, the drug is a composition of an inhibitor of NFATc3 and at least one chemotherapeutic drug, and the chemotherapeutic drug includes but is not limited to paclitaxel, doxorubicin or cisplatin.

[0075] Among them, the gene editing tool is at least one of the CRISPR-Cas9 system, shRNA or siRNA targeting the NFATc3 gene.

[0076] In some other specific embodiments, the present invention provides a gene component for conditional gene expression, comprising the following functional elements (connected in sequence from 5' to 3'):

[0077] CAG constitutive promoter, first loxP site, PGK-Neo resistance screening marker,

[0078] 6*SV40 polyadenylation signal, second loxP site, Kozak sequence,

[0079] Human NFATC3 protein coding sequence (CDS), rabbit β-globin (rBG) polyadenylation signal.

[0080] In some other specific embodiments, the present invention provides a vector targeting integration into the ROSA26 locus, comprising the above gene component and homologous arm sequences of the first intron of the ROSA26 gene located on both sides of the gene component. Among them, the length of the homologous arm is 1.5 - 5 kb.

[0081] In some other specific embodiments, the present invention provides a CRISPR-Cas9 system targeting the ROSA26 gene.

[0082] Example 2

[0083] This example provides a method for using the NFATc3 expression level as a biomarker for AAD diagnosis or prognosis assessment, and the steps are as follows:

[0084] S1 Collect aortic tissue samples from AAD patients and healthy controls;

[0085] S2 Detect the mRNA and protein expression levels of NFATc3 in the aortic tissue samples by qPCR and immunohistochemistry;

[0086] S3 When the expression level in AAD patients is higher than the healthy control threshold, it is determined as high-risk or advanced stage of AAD.

[0087] Example 3

[0088] This example provides a method for verifying the efficacy of carbamimidquine. Carbamimidquine (20 mg / kg) was intraperitoneally injected daily in an AAD mouse model. The pathological changes of the aorta were observed and the phosphorylation level of eEF2 was detected.

[0089] The results showed that carbamimidquine significantly inhibited NFATc3-induced VSMC phenotypic transformation (p<0.01).

[0090] Example 4

[0091] This example provides a method for screening compounds against aortic dissection and aneurysm, and the steps are as follows:

[0092] S1 Bind NFATc3 and the eEF2 phosphorylation site in vitro;

[0093] The method for in vitro binding is: using fluorescence resonance energy transfer (FRET) technology to detect the binding activity;

[0094] S2 Determine the inhibition rate of the compound to be screened on the binding;

[0095] S3 Screen out compounds with an inhibition rate greater than 50% for animal model verification.

[0096] Example 5

[0097] This example provides a gene therapy vector, which includes: an shRNA sequence targeting NFATc3 and a VSMC targeting delivery element.

[0098] Example 6

[0099] This example experimentally verified that the expression of NFATc3 in vascular smooth muscle cells increases during the progression of AAD.

[0100] 1. Expression of NFATc3 in aortic vascular smooth muscle cells of human thoracic aortic aneurysm: To verify whether the expression of NFATc3 is related to aortic aneurysm, the data in the single-cell transcriptome sequencing GEO database (GSE155468) from normal ascending aorta tissues or patients diagnosed with thoracic aortic aneurysm (TAA) were reanalyzed, and eight major cell types were annotated according to classical cell markers. Random sampling analysis showed that the expression of NFATc3 in SMCs was significantly upregulated in the TAA group.

[0101] 2. Expression of NFATc3 in aortic vascular smooth muscle cells of human thoracic aortic aneurysm: To verify whether the expression of NFATc3 is related to aortic aneurysm, this example analyzed the data in the single-cell transcriptome sequencing GEO database (GSE155468) from normal ascending aortic tissue or patients diagnosed with thoracic aortic aneurysm (TAA), and annotated eight major cell types according to classic cell markers. Random sampling analysis showed that the expression of NFATc3 in SMCs was significantly upregulated in the TAA group.

[0102] 3. Expression changes of NFATc3 in aortic tissues of AAD patients: Consistent with the results of the GEO database, qPCR results showed that the mRNA level of NFATc3 was significantly increased in the TAD aortic tissues of patients undergoing ascending aortic replacement surgery. WB results showed that the protein expression level of NFATc3 was significantly increased in human TAD and AAA aortic tissues. CFE results showed that the expression of NFATc3 was significantly increased in the cytoplasm and nucleus of human TAD and AAA tissues. Immunofluorescence (IF) results showed that compared with the aortic tissues of the control group, the expression of α-smooth muscle actin (ACTA2) was decreased while the expression of NFATc3 was increased in human TAD and AAA tissues.

[0103] (3) Expression changes of NFATc3 in murine AAD

[0104] In the BAPN / AngII and AAV-PCSK9 DY / AngII mouse models, WB results showed that the protein level of NFATc3 was significantly increased compared with the control group. IF results showed that compared with the aortic tissues of the control group, the expression of ACTA2 was decreased while the expression of NFATc3 was significantly increased.

[0105] Example 7

[0106] This example provides a method for constructing vascular smooth muscle cell-specific NFATc3 knockout mice, and the method is as follows:

[0107] By using CRISPR-Cas9 technology, loxP sites were inserted at both ends of exons 2 and 3 of the NFATc3 gene in the C57BL / 6J mouse background. Two gRNAs (gRNA1: 5′-GTCTTTACGAACAGTAAACC-3′; gRNA2: 5′-GTACTCGCCCTTTATGTATG-3′) were synthesized and cloned into the gRNA expression vector. After mixing this vector with Cas9 mRNA and single-stranded donor DNA, it was introduced into fertilized eggs of C57BL / 6J mice by microinjection. After confirming that the two introduced LoxP sites were inserted at the expected sites in the mice by PCR, the offspring were mated with heterozygous NFATc3 fl+ / - mice to obtain homozygous NFATc3 fl / fl mice. By mating NFATc3 fl / fl mice with Myh11-Cre mice and using tamoxifen to induce Cre expression, vascular smooth muscle cell-specific NFATc3 knockout mice (NFATc3 smcKO ) were obtained. The genotype of the offspring mice was identified by amplifying the designated primer pairs. All animal experiments were conducted in accordance with the animal research policy of Nanfang Hospital, Southern Medical University (approval number NFYY-2021-0731) and complied with the relevant regulations of the guidelines for the use and care of laboratory animals in China.

[0108] Establishment of the AAD model: Tamoxifen (20 mg / mL) was dissolved in corn oil and intraperitoneally injected into 3-week-old male NFATc3 smcKO or NFATc3 fl / fl mice at a dose of 75 mg / kg per day for five consecutive days. And at the fifth week, water containing β-aminopropionitrile (BAPN, 0.3 g / kg / day) was given for 28 days, followed by subcutaneous implantation of a slow-release pump containing angiotensin II (AngII, 1000 ng / kg / min) for 3 days of infusion ( Figure 1-6 A).

[0109] Establishment of the AAA model: Tamoxifen (20 mg / mL) was dissolved in corn oil and intraperitoneally injected into 6-week-old male NFATc3 smcKO or NFATc3 fl / fl mice at a dose of 75 mg / kg per day for five consecutive days. And at the 8th week, AAV-PCSK9 DY (2×10 11Genomic copies), and then a high-fat diet (40% fat, 11.4% sucrose, 1.25% cholesterol) was given for 6 weeks. At the 10th week, a slow-release pump containing Ang II (1500 ng / kg / min) was implanted subcutaneously in the mice for 28 days of infusion.

[0110] Establishment of the TAAD model: SPF-grade 5-week-old male NFATc3 smcKO or NFATc3 fl / fl mice were selected and randomly divided into two groups. One group was given normal saline, and the other group was given water containing β-aminopropionitrile (BAPN, 0.3 g / kg / day) for 28 days. The laboratory environment was maintained at a temperature range of 20 - 25 °C, and the relative humidity was maintained at 50% - 60%. A 12-hour light cycle was implemented to simulate the circadian physiological rhythm. All experimental animals were housed in the SPF-level animal laboratory of the Animal Experiment Center of Nanfang Hospital, Southern Medical University, and approval from the Animal Ethics Committee of Nanfang Hospital, Southern Medical University, was obtained. Figure 1 It is a schematic diagram for the construction of vascular smooth muscle cell-specific NFATc3 knockout mice.

[0111] Example 8

[0112] In this example, experiments were conducted using the model of Example 7 to verify that vascular smooth muscle cell-specific NFATc3 knockout inhibits the occurrence of AAD.

[0113] The results of the aortic morphology diagrams showed that in the saline group, there was no obvious difference in the aortic morphology between NFATc3 smcKO and NFATc3 fl / fl mice ( Figure 2 A). However, in the BAPN / AngII group, compared with NFATc3 fl / fl mice, the knockout of NFATc3 in VSMCs could inhibit the occurrence of AAD, showing a lower mortality rate and AAD incidence rate ( Figure 2 A - D). In addition, compared with NFATc3 fl / fl mice, the thoracic aortic diameter of NFATc3 smcKO mice was significantly reduced ( Figure 2 E). VSMC-NFATc3 knockout could also reduce the degradation of elastin and collagen ( Figure 2 F - H).

[0114] Figure 2 It is the experimental result diagram showing that VSMC-NFATc3 knockout can alleviate the occurrence of AAD in mice

[0115] A - H, for 5-week-old NFATc3 fl / fl or NFATc3smcKO Male mice were fed with BAPN (0.3 g / kg / d) for 28 days, and then infused with AngII (1000 ng / kg / min) for 3 days (n = 22). A, Morphological map of the mouse aorta. B, Echocardiogram of the thoracic aorta. C, Survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. D, Incidence of aortic dissection in each group. E, Maximum diameter of the aorta (NFATc3 fl / fl n = 15; NFATc3 smcKO n = 22). F, H&E staining, van Gieson staining (elastin), and Masson staining of the mouse aorta. G-H, Degradation grade of elastin (G) and collagen content (H) in the aortic wall.

[0116] In the PCSK9 DY / AngII-induced AAA model, compared with NFATc3 fl / fl mice, knockout of NFATc3 in VSMCs could prevent the occurrence of AAA, showing a lower mortality rate and incidence of AAA ( Figure 3 A-D). In addition, compared with NFATc3 fl / fl mice, the abdominal aortic diameter of NFATc3 smcKO mice was significantly reduced ( Figure 3 E). VSMC-NFATc3 knockout could also reduce the degradation of elastin and collagen ( Figure 3 F-H).

[0117] Figure 3 Experimental result graph showing that VSMC-NFATc3 knockout can alleviate the occurrence of AAA in mice, Figure 3 In Figures A-H, 8-week-old NFATc3 fl / fl or NFATc3 smcKO mice were injected with AAV-PCSK9 DY via the tail vein and fed a high-fat diet. Two weeks later, AngII (1500 ng / kg / min) infusion was started for 4 weeks (NFATc3 fl / fl n = 20; NFATc3 smcKO n = 21). A, Morphological map of the mouse aorta. B, Echocardiogram of the abdominal aorta. C, Survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. D, Incidence of abdominal aortic aneurysm in each group. E, Maximum diameter of the aorta (NFATc3 fl / fl n = 17; NFATc3 smcKOn = 21). F, H&E staining, van Gieson staining (elastin), and Masson staining of mouse aorta. G-H, Elastin degradation grade (G) and collagen content (H) of aortic wall.

[0118] 3. Effects of Vascular Smooth Muscle Cell-Specific NFATc3 Knockout on Body Weight and Four Serum Lipid Parameters

[0119] In the AAD model, NFATc3 knockout can reduce systolic blood pressure, while body weight and serum lipids show no significant changes.

[0120] In the AAA model, NFATc3 knockout can reduce systolic blood pressure, while body weight and serum lipids show no significant changes.

[0121] 4. VSMC-NFATc3 Can Aggravate the Development of TAD

[0122] Overexpression of VSMC-NFATc3 promotes the development of AAD and AAA in mice;

[0123] In the AAD and AAA models, NFATc3 overexpression can increase systolic blood pressure, while body weight and serum lipids show no significant changes.

[0124] 5. VSMC-NFATc3 Promotes the Development of the TAAD Model Induced by BAPN; Under the BAPN Modeling Method, VSMC-NFATc3 Does Not Affect Blood Pressure, Body Weight, and Serum Lipid Levels.

[0125] Example 9

[0126] In this example, the mouse model of Example 7 was used for experiments, which verified that the increased expression of NFATc3 promotes the development of AAD through a mechanism independent of blood pressure. Specifically, VSMC-NFATc3 can increase systolic blood pressure after AngII infusion. However, in the absence of AngII infusion during the administration of BAPN alone, overexpression of VSMC-NFATc3 can exacerbate the death of mice caused by the progression of AAD disease. The increased expression of NFATc3 may promote the development of AAD through a potential mechanism independent of blood pressure, and the abnormal expression of NFATc3 is crucial for the occurrence and development of AAD.

[0127] In some other specific examples, the following conclusions were obtained through experiments in this example: Knockout of VSMC-NFATc3 can restore the vascular contraction and relaxation functions reduced by BAPN modeling ( Figure 4 A-B).

[0128] In some other specific embodiments, the following conclusions were obtained in animal experiments: The knockout of VSMC-NFATc3 reduced the phenotypic transformation of VSMCs from the contractile type to the synthetic type. After treatment with BAPN, the expression of the contractile markers ACTA2, CNN1, and SM22α in the aorta of NFATc3 fl / fl mice was significantly decreased, the expression of the synthetic marker osteopontin (OPN) in the aorta increased, and the deletion of VSMC-NFATc3 could restore the expression levels of the contractile markers (ACTA2, CNN1, and SM22α) and reduce the expression of the synthetic marker OPN ( Figure 5 ). These findings further confirmed that the deletion of NFATc3 plays a beneficial role in the pathogenesis of TAAD during VSMC phenotypic transformation. Figure 5 Result graph showing that the deletion of VSMC-NFATc3 reduces the phenotypic transformation of VSMCs from the contractile type to the synthetic type. Figure 5 Specifically shown are the protein expression and quantitative analysis results of ACTA2, CNN1, SM22α, and OPN in the aorta of mice (n = 6).

[0129] In addition, the present invention also constructed two transgenic mice, namely vascular smooth muscle cell-specific knockout of NFATc3 (NFATc3 smcKO ) mice and vascular smooth muscle cell-specific overexpression of NFATc3 (NFATc3 smcKI ) mice. The present invention observed that vascular smooth muscle cell-specific knockout of NFATc3 could significantly reduce the incidence, mortality, and maximum diameter of the aorta of AAD, while vascular smooth muscle cell-specific overexpression of NFATc3 could significantly increase the incidence, mortality, and maximum diameter of the aorta of AAD.

[0130] Example 10

[0131] In this example, by establishing an AAD model of vascular smooth muscle cell-specific NFATc3 overexpression mice, it was verified that vascular smooth muscle cell-specific NFATc3 overexpression promotes the occurrence of AAD.

[0132] The experimental results showed that the knockout of VSMC-NFATc3 in mice could significantly reduce the mortality, incidence of TAAD, and pathological dilation of the aorta induced by BAPN, while the overexpression of VSMC-NFATc3 in mice could increase the mortality, incidence of TAAD, and pathological dilation of the aorta induced by BAPN. At the same time, the overexpression of NFATc3 could also accelerate the breakage and disorder of elastic fibers, increase collagen degradation, and promote the degeneration of the aortic media layer, while the knockout of NFATc3 could reduce the breakage and disorder of elastic fibers and reduce collagen degradation.

[0133] In addition, in this embodiment, three AAD animal models, namely BAPN / Ang II, BAPN, and AAV-PCSK9 / Ang II, were established, and the present invention found that NFATc3 was upregulated in AAD tissues. DY In the / Ang II group, it was found that the expression of NFATc3 increased in AAD tissues.

[0134] Example 11

[0135] This example verified that nuclear NFATc3 upregulates the expression of MMP2 and MMP9, thereby promoting the degradation of ECM.

[0136] Animal model construction: Mice with vascular smooth muscle cell-specific knockout of NFATc3 (VSMC-NFATc3-KO) and wild-type controls (WT) were used. Some WT mice were treated with β-aminopropionitrile (BAPN, a modeling agent for inducing aortic dissection / aneurysm) as a positive control. mRNA level: The expression of MMP and MMP9 in aortic tissues was detected by qRT-PCR (primers are shown in Table 1). Western blot analysis was used to analyze the protein expression of MMP2 and MMP9.

[0137] Pathological correlation: The degree of ECM degradation (such as the loss of fibronectin and collagen) was evaluated by histological analysis.

[0138] 2. Experimental results

[0139] NFATc3 knockout inhibits MMP2 / 9 expression: In the aortic tissues of VSMC-NFATc3-KO mice compared with the WT group:

[0140] The mRNA levels of MMP2 and MMP9 decreased by 60% - 70% (P < 0.01, Figure 6 B); the protein expressions of MMP2 and MMP9 were significantly reduced ( Figure 6 C - D).

[0141] BAPN-induced upregulation of MMP2 / 9 depends on NFATc3: In WT mice treated with BAPN, the expressions of MMP2 and MMP9 were increased by 2.5 - 3.0 times compared with the untreated group (P < 0.001, Figure 6 C - D);

[0142] In VSMC-NFATc3-KO mice, the promoting effect of BAPN on MMP2 / 9 expression was significantly inhibited (increase < 50%, P < 0.05).

[0143] Overexpression of NFATc3 exacerbates MMP2 / 9 expression: In mice with VSMC-specific overexpression of NFATc3, the mRNA and protein levels of MMP2 and MMP9 were increased by 2.0 - 2.5 times compared with the control group (P < 0.01).

[0144] Association between ECM degradation and disease phenotypes; NFATc3 knockout: reduced degradation of fibronectin and type I collagen (COL1) in aortic tissues, improved structural integrity of the vessel wall;

[0145] NFATc3 overexpression / BAPN treatment: exacerbated ECM degradation, accompanied by increased aortic dilation and incidence of dissection.

[0146] This example demonstrates that:

[0147] NFATc3 is a key positive regulator of MMP2 and MMP9 expression, and its deletion can significantly inhibit the transcription and translation of MMP2 / 9; pathological stimuli (such as BAPN) upregulate MMP2 / 9 through the NFATc3-dependent pathway, thereby driving ECM degradation; targeted inhibition of the NFATc3-MMP2 / 9 axis can alleviate ECM damage, providing a new strategy for the treatment of aortic dissection / aneurysm.

[0148] Figure 6 This is the experimental result graph of Example 11 of the present invention demonstrating that NFATc3 can upregulate the expression of MMP2 and MMP9 in vivo.

[0149] Figure 6 In A, after treatment with BAPN, the gene expression profiles of NFATc3 smcKO mice were compared with those of NFATc3 fl / fl mice, and a heat map was drawn based on the genes with significantly different expression related to ECM tissues (blue indicates downregulation; red indicates upregulation). Genes with a P value < 0.05 were considered statistically significant. B, relative mRNA levels of genes related to ECM tissues in the aortas of NFATc3 smcKO and NFATc3 fl / fl mice treated with BAPN (n = 6). C, protein expression levels of MMP2 and MMP9 in the aortas of NFATc3-KI fl / fl and NFATc3 smcKI mice treated with control or BAPN. D, protein expression levels of MMP2 and MMP9 in the aortas of NFATc3 smcKO and NFATc3 fl / fl mice treated with control or BAPN (n = 6).

[0150] In this example, further experiments on this transcriptional regulation in VSMCs were carried out, and the results of nuclear transcriptional activity assays showed that NFATc3 upregulated the transcriptional levels of MMP9 and MMP2 ( Figure 7 A - B). Figure 7 In which NFATc3 can upregulate the transcriptional levels of MMP9 and MMP2.

[0151] The present invention uses the JASPER database to search for potential NFATc3 binding sites in the MMP9 and MMP2 promoters (fragments -1.0 - 0.5 and -2.0 - 1.5 kb upstream of the transcription start site, respectively) ( Figure 8 A). The functional NFATc3 binding site is located at a position with evolutionarily conserved characteristics ( Figure 8 B - C). Figure 8 Potential binding sites of NFATc3 in the MMP2 and MMP9 promoters. Figure 8 Among them, A is the NFATc3 binding site. B - C, Schematic diagrams of the binding sites of NFATc3 on the MMP9 (B) and MMP2 (C) promoters and the positions of the evolutionarily conserved characteristics of the NFATc3 recognition sequence.

[0152] Luciferase reporter gene experiments showed that NFATc3 could increase the luciferase activity driven by the MMP9 and MMP2 promoters, and mutating the potential NFATc3 binding sites could reduce the NFATc3 - dependent effect ( Figure 9 ).

[0153] Figure 9 For NFATc3 to up - regulate the luciferase activity driven by the MMP9 and MMP2 promoters, and the mutation of the NFATc3 binding site to down - regulate the NFATc3 - dependent effect. Luciferase reporter gene analysis in 293T cells (n = 6).

[0154] Chromatin immunoprecipitation quantitative PCR experimental results showed that NFATc3 was enriched in H3K27ac (enhancer and promoter marker) and H3K4me3 (promoter - specific marker) in VSMCs, and these markers were located proximal to the MMP9 and MMP2 promoters ( Figure 10 ). Figure 10 For NFATc3 to be enriched in H3K27ac and H3K4me3 proximal to the MMP9 and MMP2 promoters in VSMCs.

[0155] ChIP - qPCR verified the enrichment of H3K27ac and H3K4me3 on the MMP9 and MMP2 promoters (n = 6).

[0156] The above results indicate that VSMC - NFATc3 can promote MMP activation and ECM degradation, and NFATc3 can directly target and transcriptionally up - regulate the expression levels of MMP9 and MMP2.

[0157] In addition, experiments showed that NFATc3 smcKO increased the expression of fibronectin and type I collagen in the lesion area of the mouse aorta

[0158] Example 12

[0159] This example verified that cytoplasmic NFATc3 promotes the phenotypic transformation of VSMCs by increasing the protein synthesis rate in ribosomes.

[0160] This example showed through experiments that:

[0161] During the progression of AAD, the expression of NFATc3 in the cytoplasm and nucleus of VSMCs from the aorta increased;

[0162] The results of LC-MS / MS analysis suggested that cytoplasmic NFATc3 was closely related to ribosome function;

[0163] The polysome technique revealed that NFATc3 bound to the large and small subunits of ribosomes;

[0164] NFATc3 was able to increase the protein synthesis rate;

[0165] NFATc3 was able to increase the synthesis of nascent proteins;

[0166] Increasing the protein synthesis rate could promote the phenotypic transformation of vascular smooth muscle cells from the contractile type to the synthetic type;

[0167] Overexpression of NFATc3 could inhibit the expression of contractile marker proteins of VSMCs induced by PDGF-BB and increase the expression of synthetic marker proteins, and this result could be reversed after treatment with CHX ( Figure 11 A). Deletion of NFATc3 could increase the expression of contractile marker proteins of VSMCs induced by TGF-β and decrease the expression of synthetic marker proteins, and this result could be reversed after treatment with Y-320 ( Figure 11 B). Downregulation of NFATc3 also increased the expression of contractile marker proteins of VSMCs induced by PDGF-BB in HASMCs ( Figure 11 C). Figure 11 NFATc3 can promote the phenotypic transformation of vascular smooth muscle cells from the contractile type to the synthetic type.

[0168] Figure 11 In, A, Immunoblotting results and quantitative analysis (n = 4) of ACTA2, CNN1, SM22α, and OPN after treatment with CHX (50 μM) for 12 hours and PDGF-BB (20 μg / L) for 24 hours in MASMCs isolated from the aortas of NFATc3-KI fl / fl and NFATc3 smcKI mice. B, From NFATc3 fl / fl and NFATc3 smcKOMASMCs isolated from mouse aorta, after being treated with TGF-β (10 μg / L) for 48 hours and then with Y-320 (2 μM) for 24 hours, immunoblotting results and quantitative analysis of ACTA2, CNN1, SM22α and OPN (n = 4). C, HASMCs were transfected with siR-Con or siR-NFATc3 for 48 hours and then treated with PDGF-BB for 24 hours, immunoblotting results and quantitative analysis of ACTA2, CNN1, SM22α and OPN (n = 4).

[0169] The results of co-immunoprecipitation analysis showed that NFATc3 interacted with RPL28 and RPS6, indicating that NFATc3 was directly localized in ribosomes. Therefore, the experimental results of this example showed that cytoplasmic NFATc3 promoted translation in ribosomes and increased the protein synthesis rate, thus promoting the phenotypic switch of VSMCs from the contractile phenotype to the synthetic phenotype. NFATc3 was localized in ribosomes. Immunoprecipitation of MASMC lysates was performed using an anti-NFATc3 antibody. The expression of RPL28 and RPS6 was detected by immunoblotting.

[0170] Example 13

[0171] This example verified that NFATc3 promoted the protein synthesis rate by inhibiting the phosphorylation of eukaryotic elongation factor 2 (eEF2).

[0172] Given that NFATc3 plays a key role in the VSMC phenotypic switch mediated by protein synthesis, the present invention studied its potential molecular mechanism. The present invention extracted known proteins that interact with NFATc3, including 11 40S ribosomal proteins, 19 60S ribosomal proteins, 5 elongation factors and 1 initiation factor, which were significantly enriched during translation. NFATc3 was directly localized in ribosomes and might play a key role during elongation. The activity of eEF2 was regulated by eEF2K, which phosphorylated eEF2 (p-eEF2) at Thr56 and inhibited elongation, while eEF2 could be dephosphorylated by protein phosphatase 2A (PP2A) to promote protein synthesis.

[0173] This example further verified the potential interaction between NFATc3 and eEF2. The results of protein co-immunoprecipitation proved that NFATc3 could bind to non-phosphorylated eEF2, rather than phosphorylated eEF2 (p-eEF2) ( Figure 12 ). Figure 12 In, immunoprecipitation of MASMC lysates was performed using an anti-NFATc3 antibody; immunoblot analysis of the precipitate was performed using anti-eEF2 and anti-phosphorylated eEF2 antibodies.

[0174] This example further verified that the knockout of VSMC-NFATc3 had little effect on the overall eEF2 protein expression level, but it increased the protein level of p-eEF2 in mouse aortic tissues after treatment with control or BAPN( Figure 13 ). Figure 13 In fl / fl and smcKO mice, they were treated with control or BAPN for 28 days respectively, and then immunoblotting and quantitative analysis of p-eEF2 and eEF2 in their aortic tissues were performed (n = 4).

[0175] In addition, NFATc3 did not affect the expression levels of eEF2K or PP2A proteins. Therefore, the knockout of VSMC-NFATc3 did not change the expression of eEF2K or PP2A, and the increased p-eEF2 level was not due to the change in the activity of eEF2K or PP2A. NFATc3 was directly localized on ribosomes and might have an elongation function. Therefore, the present invention speculated that the decrease in the binding of NFATc3 to the eEF2 phosphorylation site led to the change in the p-eEF2 level.

[0176] This example constructed two mutant plasmids, which expressed eEF2 T56A and T56D proteins respectively to simulate non-phosphorylated eEF2 and phosphorylated p-eEF2, so as to determine whether the binding of NFATc3 affected the phosphorylation of eEF2.

[0177] The affinity of Flag-NFATc3 for T56A was significantly higher than that for T56D ( Figure 14 ), confirming that NFATc3 might directly bind to non-phosphorylated eEF2 around Thr56. Figure 14 NFATc3 preferentially binds to non-phosphorylated eEF2. Immunoblot experiment of NFATc3 preferentially binding to proteins. After transfection of eEF2-T56D and eEF2-T56A plasmids in HEK293T cells for 48 hours, immunoprecipitation of the lysates was performed with anti-NFATc3 antibody (n = 4).

[0178] To further identify which domain of NFATc3 binds to eEF2, the present invention constructed pCMV6-Flag plasmids containing the full-length NFATc3 and three NFATc3 domains (NFAT homologous region (NHR, amino acids 1–415), REL homologous region (RHR, amino acids 416–597), and C-terminal region (amino acids 598–1076)). Before performing co-immunoprecipitation, the present invention transfected the His-eEF2 plasmid into HEK293T cells and incubated with a Flag antibody as the primary antibody. The results showed that the full-length NFATc3 and the RHR domain could specifically bind to His-eEF2, while the NHR or C-terminal domain could not bind to His-eEF2. The present invention also found that the deletion of the NFATc3 RHR domain could eliminate the interaction between NFATc3 and eEF2( Figure 15 A). Mammalian two-hybrid assays showed that the RHR domain bound to eEF2( Figure 15 B), indicating that NFATc3 could directly bind to eEF2. Figure 15 NFATc3 can bind to eEF2 through its RHR domain.

[0179] Figure 15 In, A, Schematic diagram of the NFATc3 structure and the binding between NFATc3 and eEF2 are indicated by + or - (top schematic diagram). Co-transfect the full-length His-eEF2 and various truncated Flag-NFATc3 plasmids into 293T cells, perform co-immunoprecipitation using an anti-Flag antibody, and then perform immunoblot analysis (bottom schematic diagram). B, Mammalian two-hybrid analysis of the interaction between NFATc3 and eEF2. Co-transfect the pBIND plasmids encoding the full-length NFATc3, 1-415aa, 416-597aa, and 598-1076aa domains and the pACT plasmid expressing the full-length eEF2 into COS-7 cells, and measure the luciferase activity 48 hours after transfection.

[0180] In addition, in MASMCs, the deletion of the NFATc3 RHR domain eliminated the interaction that down-regulated the phosphorylation level of eEF2 induced by NFATc3.

[0181] Immunofluorescence results showed that compared with the aortic tissue of the control group, the expression level of eEF2 in the aortic tissue of TAD patients increased, while the expression level of phosphorylated eEF2 decreased.

[0182] Overexpression of eEF2 reversed the expression of TGF-β-induced VSMC contractile markers and the inhibition of synthetic markers. Therefore, eEF2 is closely related to the phenotypic switch from contractile to synthetic of NFATc3.

[0183] To clarify the role of p-eEF2 / eEF2 in NFATc3-mediated protein synthesis and the transition from a contractile to a synthetic phenotype, the present invention used A484954 to treat MASMCs isolated from NFATc3 fl / fl and NFATc3 smcKO aortas. A484954 is a selective eEF2K inhibitor that increases the activity of eEF2. The research results found that treatment with A484954 could reverse the decrease in protein synthesis rate observed in VSMC-NFATc3 deficiency, and treatment with cabamiquine reversed the enhancement effect observed in NFATc3 smcKI VSMCs. Cabamiquine inhibits the activity of eEF2 and increases phosphorylation ( Figure 16 A-B). Figure 16 NFATc3 increases the protein synthesis rate by inhibiting the phosphorylation of eEF2. Figure 16 In A, after MASMCs of NFATc3 fl / fl and NFATc3 smcKO mice were treated with control or A484954 (10 μM, 10 min), puromycin incorporation experiments were performed on them, and the expression levels of p-eEF2 / eEF2 were detected by immunoblotting (n = 4). B, after MASMCs of NFATc3-KI fl / fl and NFATc3 smcKI mice were treated with control or cabamiquine (2 μM, 12 h), puromycin incorporation experiments were performed on them, and the expression levels of p-eEF2 / eEF2 were detected by immunoblotting (n = 4).

[0184] Meanwhile, treatment with A484954 reduced the expression of contractile marker proteins in VSMCs caused by NFATc3 deficiency, while cabamiquine reversed the inhibitory effect of NFATc3 on VSMC contractile markers in VSMCs of NFATc3 smcKI mice. Therefore, NFATc3 deficiency affects the binding of eEF2 to translating ribosomes, increases the phosphorylation of eEF2, and inhibits translation elongation, resulting in a decrease in protein synthesis rate and ultimately inhibiting the phenotypic transition of VSMCs from contractile to synthetic. Increasing or decreasing eEF2 activity can affect the phenotypic transition of vascular smooth muscle cells

[0185] In the TAAD model of mice, the present invention established the relationship between NFATc3 and eEF2 and used cabamiquine to further explore potential therapeutic targets of NFATc3 in TAAD. Specifically, in this part of the experiment, the present invention used NFATc3 smcKOA BAPN-induced TAAD model was constructed in mice and treated with Lenti-Con or Lenti-eEF2 respectively. Increased expression levels of eEF2 were observed in MASMCs treated with Lenti-eEF2. The use of Lenti-eEF2 could increase the occurrence of BAPN-induced TAAD in mice. This invention was experimentally proven that Lenti-eEF2 could promote the occurrence of TAAD, which was verified through the following experimental aspects: A, experimental design of the BAPN model. B, immunoblotting and quantitative analysis showing eEF2 expression in MASMCs (n = 6). C, morphology of the mouse aorta. D, incidence of TAAD. E, survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. F, maximum diameter of the aorta. smcKO In this example, it was verified that carbamimidazole could reduce the probability of AAD occurrence.

[0186] Example 14

[0187] This example verified that carbamimidazole could reduce the probability of AAD occurrence.

[0188] This invention also conducted experiments on NFATc3 smcKI mice treated with saline or carbamimidazole ([A]). In the control group, the morphology of the aorta in NFATc3 Figure 17 mice did not change under the conditions of receiving saline or carbamimidazole treatment; however, under the condition of receiving BAPN treatment, carbamimidazole could inhibit the occurrence of BAPN-induced TAAD in NFATc3 smcKI mice ([B]). In NFATc3 smcKI mice treated with carbamimidazole, the incidence and mortality of TAAD were significantly reduced, and the aortic diameter was smaller than that of NFATc3 Figure 17 mice treated with saline ([C - E]). Carbamimidazole could also reduce the degradation of elastin and collagen ([F - H]). Carbamimidazole could reduce the probability of TAAD occurrence. smcKI mice. The incidence and mortality of TAAD in NFATc3 smcKI mice treated with carbamimidazole were significantly reduced, and its aortic diameter was smaller than that of NFATc3 Figure 17 mice treated with saline ([C - E]). Carbamimidazole could also reduce the degradation of elastin and collagen ([F - H]). Carbamimidazole could reduce the probability of TAAD occurrence. Figure 17 Carbamimidazole could reduce the probability of TAAD occurrence.

[0189] Figure 17In A, schematic diagram of the experimental design of the BAPN model. In B, morphological diagram of the mouse aorta. In C, TAAD incidence rate. In D, survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. In E, maximum diameter of the aorta. In F, H&E staining, van Geison (elastin) staining, and Masson staining of the mouse aorta. In G-H, degree of elastin degradation (G) and collagen content (H) in the aortic wall. The present invention also found that carbamimidazole has no effect on body weight, blood pressure, and blood lipid levels.

[0190] Carbamimidazole increased the ratio of p-eEF2 / eEF2 in the aorta of NFATc3 smcKI mice treated with BAPN, which is consistent with the results of the in vitro cell experiment research of the present invention. In addition, carbamimidazole also inhibited the phenotypic transformation of VSMCs from the contractile type to the synthetic type caused by overexpression of NFATc3. Including the reduction of AAD incidence, mortality, and aortic diameter ( Figure 18 A-E). Figure 18 Experimental result diagram showing that carbamimidazole can reduce the occurrence of AAD.

[0191] Figure 18 In A, morphological diagram of the aorta. In B, AAD incidence rate. In C, survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. In D, maximum aortic diameter (saline: n = 6; carbamimidazole: n = 12). The measurement results do not include mice that died due to aortic rupture. In E, schematic diagram of the establishment of the BAPN / AngII model.

[0192] Under this modeling method, treatment with carbamimidazole did not affect NFATc3 smcKI in the body weight, blood pressure, or blood lipid levels of mice.

[0193] In the PCSK9 DY / AngII-induced mouse AAA model, carbamimidazole can inhibit the occurrence of AAA caused by overexpression of NFATc3, including the reduction of AAA incidence, mortality, and aortic diameter ( Figure 19 A-E). Figure 19 Carbamimidazole can reduce the probability of the occurrence of AAA.

[0194] Figure 19 In A, morphological diagram of the aorta. In B, AAA incidence rate. In C, survival curve obtained by Kaplan-Meier analysis and compared using the log-rank test. In D, maximum aortic diameter (saline: n = 6; carbamimidazole: n = 12). The measurement results do not include mice that died due to aortic rupture. In E, AAV-PCSK9 DY / Schematic diagram for the establishment of the AngII model.

[0195] In the AAA model, treatment with carbamimidazole did not affect the levels of NFATc3 smcKI body weight, blood pressure or blood lipids in mice.

[0196] In addition, increased protein expression levels of p-eEF2 were observed in NFATc3 smcKI mice treated with carbamimidazole ( Figure 20 ). These findings indicate that carbamimidazole inhibits the occurrence of AAD in a NFATc3-dependent manner and protects the aorta from degeneration, dissection and rupture. Carbamimidazole can increase the expression of p-eEF2 in NFATc3 smcKI mice.

[0197] Figure 20 In smcKI NFATc3 DY mice, after treatment with AAV-PCSK9

[0198] / AngII or saline, immunoblotting and quantitative analysis of p-eEF2 and eEF2 in aortic tissues (n = 5). 2+ i In summary, the present invention used three AAD models to clarify the effect of NFATc3 on AAD. BAPN is commonly used to induce aortic wall rupture and dilation by disrupting the cross-linking of procollagen and tropoelastin and damaging the aortic wall integrity. The combination of BAPN and AngII can induce aortic AAD with a higher incidence of rupture. AAV carrying PCSK9 D377Y (Asp374-to-Tyr mutant) is used to induce hyperlipidemia by inducing the degradation of low-density lipoprotein receptors in hepatocytes, and combined with AngII infusion and high-fat diet to induce AAA. Knockout of VSMC-NFATc3 significantly slowed down the occurrence and development of AAD, while its overexpression accelerated the occurrence and development of AAD in VSMC-NFATc3 knock-in mice. In addition, NFATc3 increased the blood pressure level in mice under AngII stimulation. This is contradictory to previous findings related to VSMCs. Sustained

[0199] ​The present invention reveals a new function of NFATc3 in regulating protein synthesis. It interacts with eEF2 at Thr56 and inhibits its phosphorylation in ribosomes, without affecting the expression of eEF2K or PP2A. In addition, activating eEF2 with A-484954 reversed the slowdown of protein synthesis rate and VSMC phenotypic transformation caused by NFATc3 deficiency. More importantly, the upregulation of eEF2 reversed the protective effect in the progression of AAD caused by NFATc3 deficiency. Conversely, cabamiquine (an inhibitor of eEF2) reversed the increased protein synthesis and VSMC phenotypic transformation caused by NFATc3 overexpression. NFATc3 binds to eEF2 through the RHR, i.e., the DNA-binding domain, reducing the phosphorylation level of eEF2, thereby increasing its activity to promote protein synthesis. These findings indicate that NFATc3-mediated regulation is crucial for VSMC phenotypic transformation and protein synthesis.

[0200] Cabamiquine, an antimalarial drug, targets eEF2 and inhibits protein synthesis to combat malaria parasites. Due to its effectiveness and safety, it is currently in phase I clinical trials for the treatment of Plasmodium falciparum sporozoites (https: / / clinicaltrials.gov; NCT04250363, NCT03261401). In the invention, supplementing cabamiquine significantly slowed down the phenotypic transformation of VSMCs in a NFATc3-dependent manner and inhibited the progression of the AAD model induced by BAPN, BAPN / AngII, and PCSK9 DY / AngII. The in vivo animal experimental research results of the present invention provide a new direction for cabamiquine to prevent and treat AAD by pharmacologically inhibiting eEF2.

[0201] Example 15

[0202] This example provides a drug for treating aortic dissection and aneurysm, which is a drug that inhibits eEF2.

[0203] In some other specific embodiments, the drug that inhibits eEF2 is cabamiquine. Compounds targeting eEF2 include, but are not limited to, cabamiquine or / and its pharmaceutically derivatives, eEF2 kinase inhibitors or / and their pharmaceutically derivatives.

[0204] In some other specific embodiments, a pharmaceutical composition for treating aortic dissection and aneurysm is an eEF2 kinase inhibitor, a natural extract, an excipient, and a solvent.

[0205] More specifically, in the composition: when the solvent of the eEF2 kinase inhibitor is NH125, its content is 10 - 50 mg / unit dose;

[0206] Excipients: microcrystalline cellulose is 50 - 200 mg, and magnesium stearate lubricant is 1 - 5 mg;

[0207] The pharmaceutical composition provided by this embodiment has a better effect in treating aortic dissection and aneurysm.

[0208] Table 1 Design of animal experiments

[0209]

[0210]

[0211] Table 2 Primers used in the present invention

[0212]

[0213]

[0214] Table 4 Chinese - English abbreviation table

[0215]

[0216]

[0217]

[0218] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a drug that inhibits eEF2 in drugs for aortic dissection and aneurysm.

2. Use of the drug for inhibiting eEF2 in the drugs for aortic dissection and aneurysm, characterized in that, The drug that inhibits eEF2 includes carboquone.

3. Use of NFATc3 as a drug target for aortic dissection and aneurysm.

4. The application of NFATc3 as a drug target for aortic dissection and aneurysm according to claim 3, characterized in that: The drug is an inhibitor of NFATc3.

5. Use of NFATc3 as a drug target for aortic dissection and aneurysm according to claim 4, characterized in that: The inhibitor exerts its effect by inhibiting the expression and / or function of NFATc3 in vascular smooth muscle cells.

6. The use of NFATc3 as a drug target for aortic dissection and aneurysm according to claim 3, wherein: The drug is a compound that blocks the interaction between NFATc3 and eEF2; Optionally, the drug specifically binds to the phosphorylation site of eEF2; Optionally, the phosphorylation site of eEF2 is Thr56.

7. Use of NFATc3 as a drug target for aortic dissection and aneurysm according to claim 3, characterized in that: The drug is a compound that regulates the transcription of matrix metalloproteinases MMP9 and MMP2.

8. Use of the NFATc3 expression level as a biomarker for the diagnosis or prognosis assessment of aortic dissection and aneurysm.

9. A screening method for anti-aortic dissection and aneurysm compounds, characterized in that, It includes the following steps: S1 Bind the NFATc3 and the eEF2 phosphorylation site in vitro; S2 Determine the inhibition rate of the compound to be screened on the binding; S3 Screen out the compounds with an inhibition rate greater than 50% for animal model verification.

10. A gene therapy vector, characterized in that, It includes: An shRNA sequence targeting NFATc3 and a VSMC targeting delivery element.

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