Application of FKBP10 in diagnosis and treatment of atherosclerosis

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of FKBP10 in diagnosis and treatment of atherosclerosis. Specifically, the invention demonstrates that FKBP10 promotes the unique effect of EndMT in vascular pathology, which is associated with increasing the phenotypes of atherosclerosis and unstable plaques. The invention proves that FKBP10 promotes membrane localization of PARP1, activates PI3K / AKT and MAPK signal pathways, induces EndMT and promotes atherosclerosis and poor prognosis of patients. Meanwhile, EndMT is inhibited by screening a small molecule drug TAK733, and a new scheme is provided for clinical treatment of atherosclerosis, so that the application has good potential practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of FKBP10 in the diagnosis and treatment of atherosclerosis. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Stroke is the second leading cause of death globally and the main cause of disability in adults worldwide. The progression of carotid artery atherosclerosis (CAS) is closely related to the occurrence of cerebrovascular ischemic events such as stroke and transient ischemic attack. Early CAS is mainly manifested by an increase in clMT, while late-stage CAS is manifested by CP and carotid artery sclerosis (CS). Although through existing treatment means, such as a combination of lipid-lowering drugs, antithrombotic drugs, antihypertensive drugs, carotid endarterectomy, carotid artery stenting, etc., there is a certain curative effect, the overall treatment effect is still not ideal, bringing a huge burden to individuals, families and the national medical system. Therefore, further studying the pathogenesis of carotid artery atherosclerosis and finding new molecular diagnosis and treatment targets has more important practical significance and theoretical value.

[0004] Atherosclerosis (AS) is a chronic inflammatory disease with a complex pathological mechanism, and various cells such as endothelial cells, macrophages, and smooth muscle cells are involved in the occurrence and development of plaques. Among them, endothelial dysfunction is an important link in the development of AS. Atherosclerosis mainly occurs at the bifurcations and bends of blood vessels with disturbed flow (d-flow), and these areas are affected by oscillatory shear stress (OSS). The action of oscillatory shear stress on endothelial cells is likely to cause endothelial injury, inhibit the production of NO by endothelial cells, and further lead to endothelial-mesenchymal transition.

[0005] Recent studies have shown that endothelial-mesenchymal transition is likely to be involved in the occurrence, development and formation of atherosclerotic plaques, affecting plaque stability. After endothelial-mesenchymal transition occurs, endothelial cells gradually lose their endothelial morphology and function, dedifferentiate into endothelial progenitor cells, and then acquire stronger migratory ability and transdifferentiate into mesenchymal-like cells. EndMT is usually accompanied by a decrease in endothelial cell markers such as VE-Cadherin, CD31, ZO-1, etc. and an increase in mesenchymal-like cell markers such as Snail, N-Cadherin, ZEB1, α-SMA, etc. Endothelial cells can be transformed into smooth muscle-like cells, fibroblast-like cells, etc. through EndMT. Aberrant expression of FKBP10 will lead to collagen-related diseases such as pulmonary fibrosis, IPF, osteogenesis imperfecta, etc. Related studies have shown that FKBP10 can promote epithelial-mesenchymal transition (EMT) of various tumor cells such as clear cell renal cell carcinoma, lung cancer, glioma, etc. through activating multiple pathways, promoting tumor proliferation, invasion and metastasis. However, whether it plays a role in the transformation of atherosclerotic endothelial cells into mesenchymal cells has not been reported. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide the application of FKBP10 in the diagnosis and treatment of atherosclerosis. Specifically, the present invention discovers through research that FKBP10 promotes the membrane localization of PARP1, activates the PI3K / AKT and MAPK signaling pathways, induces EndMT and promotes atherosclerosis and poor prognosis of patients. Based on this discovery, the present invention screens the small molecule drug TAK733 to inhibit EndMT, providing a new solution for the clinical treatment of atherosclerosis. Based on the above research results, the present invention is completed.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the application of a reagent for detecting the expression level of FKBP10 in the preparation of an atherosclerotic detection product.

[0009] Specifically, the present invention discovers through research that the expression of FKBP10 is significantly up-regulated in atherosclerotic plaques and is closely related to EndMT and plaque neovascularization, which may play a key role in enhancing plaque instability. Therefore, the atherosclerotic detection product can be used for the screening, (assistant) diagnosis, monitoring or prognostic evaluation of atherosclerosis.

[0010] The substance for detecting the expression level of FKBP10 may include reagents for detecting the expression level of the FKBP10-encoding gene based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing, and / or reagents for detecting the protein (enzyme) expression level of FKBP10 based on immunoassay methods.

[0011] Further, the products include, but are not limited to, primers, probes, (gene or protein) chips, detection kits, detection devices, equipment, etc., which are not specifically defined herein.

[0012] In a second aspect of the present invention, there is provided a system for detecting atherosclerosis, the system comprising:

[0013] An acquisition module, which is configured to: acquire the expression level of FKBP10 in a sample to be tested of a subject;

[0014] An analysis module, which is configured to: analyze and judge the disease condition of the subject according to the expression level of FKBP10 of the subject obtained by the acquisition module.

[0015] The sample to be tested may be an atherosclerotic plaque sample or an endothelial cell sample of the subject, and further may be an endothelial cell sample of the neovascularization in the atherosclerotic plaque.

[0016] The detection of atherosclerosis may specifically be the screening, (auxiliary) diagnosis, monitoring or prognosis assessment of atherosclerosis, including the detection and analysis of endothelial-mesenchymal transition and plaque neovascularization in atherosclerosis.

[0017] In a third aspect of the present invention, there is provided the use of FKBP10 as a target in the preparation and / or screening of drugs for atherosclerosis.

[0018] Further, based on the effects of a candidate drug on atherosclerosis before and after use, it is determined whether the candidate drug can be used for the prevention and / or treatment of atherosclerosis.

[0019] Further, the method for screening drugs for preventing or treating atherosclerosis includes:

[0020] 1) Treat a system expressing and / or containing FKBP10 with a candidate substance; set a control without treating with the candidate substance;

[0021] 2) After completing step 1), detect the expression level of FKBP10 in the system; compared with the control, if the expression level of FKBP10 in the system treated with the candidate substance is significantly down-regulated, the candidate substance can be used as a candidate drug for preventing or treating atherosclerosis.

[0022] The system may be a solution system, a cell system (such as endothelial cells), a tissue system, an organ system or an animal (including human and non-human animals, and the non-human animals are preferably mammals) system, which is not specifically defined herein.

[0023] The fourth aspect of the present invention provides the use of a substance that inhibits the expression of FKBP10 or reduces its activity in any one or more of the following:

[0024] (a) inhibiting the membrane translocation of PARP1 or preparing a product for inhibiting the membrane translocation of PARP1;

[0025] (b) inhibiting the activation of the PI3K / AKT and MAPK signaling pathways or preparing a product for inhibiting the activation of the PI3K / AKT and MAPK signaling pathways;

[0026] (c) inhibiting endothelial-mesenchymal transition or preparing a product for inhibiting endothelial-mesenchymal transition;

[0027] (d) a product for preventing and / or treating atherosclerosis.

[0028] Among them, the substance that inhibits the expression of FKBP10 or reduces its activity includes but is not limited to RNA interference molecules or antisense oligonucleotides targeting FKBP10, small molecule inhibitors (such as TAK733), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout, etc., which are not specifically limited herein.

[0029] The product can be a drug or an experimental reagent for non-medical use, and the experimental reagent can be used for basic research. For example, the product can be used to regulate endothelial-mesenchymal transition of endothelial cells in vivo (or in vitro), etc., so as to prepare a biological model related to endothelial-mesenchymal transition of endothelial cells, thereby laying a foundation for the mechanism research of diseases such as atherosclerosis.

[0030] According to the present invention, when the product is a drug, the drug further includes at least one pharmaceutically inactive ingredient.

[0031] The pharmaceutically inactive ingredient can be a carrier, excipient, diluent, etc. commonly used in pharmacy. At the same time, the carrier, excipient, diluent, etc. that can be included as non-drug active ingredients are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0032] The drug of the present invention can be administered into the body by known methods. For example, it can be delivered systemically by intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0033] The subject to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys and chimpanzees.

[0034] The fifth aspect of the present invention provides a method for preventing and / or treating atherosclerosis, the method comprising administering to a subject the substance that inhibits the expression of FKBP10 or reduces its activity as described above and / or the product in the above application.

[0035] Beneficial technical effects of the above one or more technical solutions:

[0036] The above technical solutions demonstrate the unique role of FKBP10 in promoting EndMT in vascular pathology, which is associated with an increased atherosclerotic and unstable plaque phenotype. Specifically, the above technical solutions demonstrate that FKBP10 promotes the membrane localization of PARP1, activates the PI3K / AKT and MAPK signaling pathways, induces EndMT and promotes atherosclerosis and poor prognosis in patients. At the same time, by screening the small molecule drug TAK733 to inhibit EndMT, a new solution is provided for the clinical treatment of atherosclerosis. Therefore, it has good potential practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0038] Figure 1 FKBP10 is highly expressed in atherosclerotic plaques in the examples of the present invention, especially in neovascularization. (A) UMAP dimensionality reduction visualization analysis was used to analyze the expression of FKBP10 in plaques at different time points. (B) Representative immunohistochemical images and statistical analysis of FKBP10 in carotid artery sections of Apoe mice on normal diet (ND) and high-fat diet (HFD) (n = 10). Scale bar = 100 μm. (C) Representative immunofluorescence images showing the expression level and location of FKBP10 in carotid artery sections of Apoe C57 mice fed with normal diet (ND) and high-fat diet (HFD) (n = 10). Scale bar = 25 μm. (D) Representative H&E, Masson, and immunohistochemical images of the expression level of FKBP10 in stable and unstable atherosclerotic plaques of human common carotid arteries (n = 10). Scale bars for H&E and Masson staining = 2.5 mm, and scale bar for immunohistochemistry = 100 μm. (E) Representative immunofluorescence images of human carotid atherosclerotic plaques. Red represents CD31, green represents FKBP10, and blue represents DAPI. (F) Umap dimensionality reduction clustering shows endothelial cell subpopulations. (G) Shows the expression levels of FKBP10 in different endothelial cells. Data are represented as mean ± SD. *P < 0.05, **P < 0.01. - / - Representative immunohistochemical images and statistical analysis of FKBP10 in carotid artery sections of Apoe mice (n = 10). Scale bar = 100 μm. (C) Representative immunofluorescence images showing the expression level and location of FKBP10 in carotid artery sections of Apoe C57 mice fed with normal diet (ND) and high-fat diet (HFD) (n = 10). Scale bar = 25 μm. (D) Representative H&E, Masson, and immunohistochemical images of the expression level of FKBP10 in stable and unstable atherosclerotic plaques of human common carotid arteries (n = 10). Scale bars for H&E and Masson staining = 2.5 mm, and scale bar for immunohistochemistry = 100 μm. (E) Representative immunofluorescence images of human carotid atherosclerotic plaques. Red represents CD31, green represents FKBP10, and blue represents DAPI. (F) Umap dimensionality reduction clustering shows endothelial cell subpopulations. (G) Shows the expression levels of FKBP10 in different endothelial cells. Data are represented as mean ± SD. *P < 0.05, **P < 0.01. - / - Representative immunohistochemical images and statistical analysis of FKBP10 in carotid artery sections of Apoe C57 mice fed with normal diet (ND) and high-fat diet (HFD) (n = 10). Scale bar = 25 μm. (D) Representative H&E, Masson, and immunohistochemical images of the expression level of FKBP10 in stable and unstable atherosclerotic plaques of human common carotid arteries (n = 10). Scale bars for H&E and Masson staining = 2.5 mm, and scale bar for immunohistochemistry = 100 μm. (E) Representative immunofluorescence images of human carotid atherosclerotic plaques. Red represents CD31, green represents FKBP10, and blue represents DAPI. (F) Umap dimensionality reduction clustering shows endothelial cell subpopulations. (G) Shows the expression levels of FKBP10 in different endothelial cells. Data are represented as mean ± SD. *P < 0.05, **P < 0.01.

[0039] Figure 2 In the embodiment of the present invention, endothelial cell-specific FKBP10 knockdown accelerates the progression of atherosclerosis.

[0040] (A) Schematic illustration of the implementation of dietary intervention and adenovirus knockout treatment in Apoe− / − mice. (B) Representative images of atherosclerotic plaques in the aortic arch and carotid arteries of the two groups and quantification of plaque area (n = 10). (C) Representative Oil Red O staining images and quantification of atherosclerotic lesions in the entire aorta of the two groups (n = 10). (D) Representative H&E staining images and quantification of atherosclerotic lesion areas in the aortic root of the two groups (n = 10). Scale bar = 500 μm. (E) Representative H&E staining images and quantification of atherosclerotic lesion areas (right) in the carotid arteries of the two groups (n = 10). Scale bar = 250 μm. (F) Representative H&E staining images and quantification of atherosclerotic lesion areas (left) in the carotid arteries of the two groups (n = 10). Scale bar = 250 μm. (G) Representative Oil Red O staining images of the aortic root of the two groups and quantification of the percentage of Oil Red O positivity (n = 10). Scale bar = 500 μm. (H) Representative Oil Red O staining images of the carotid arteries (right) of the two groups of patients and quantification of the percentage of Oil Red O positivity (n = 10). Scale bar = 250 μm. (I) Representative Masson staining images of the aortic root of the two groups and quantification of the percentage of collagen positivity (n = 10). Scale bar = 500 μm. (J) Representative Masson staining images and quantification of the percentage of collagen positivity (right) in the carotid arteries of the two groups (n = 10). Scale bar = 250 μm. (K) Immunofluorescence analysis was used to determine the level of macrophage infiltration in sections of the aortic sinus (left panel) and carotid arteries (right panel) of the two groups (n = 10), represented by the expression level of F4 / 80 (red) (n = 10). Scale bar = 100 μm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0041] Figure 3 In the embodiment of the present invention, FKBP10 silencing inhibits EndMT in vitro.

[0042] (A) qRT-PCR analysis of FKBP10 mRNA levels in HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). (B) Gene set enrichment analysis (GSEA) of pathways in HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h) showed a high enrichment of negative regulation of EMT. (C) qRT-PCR analysis of ZEB1, VE-Cadherin and Snail mRNA levels in HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and ox-LDL stimulation (100 μg / ml, 48 hours). (D) qRT-PCR analysis of FKBP10, ZEB1, VE-Cadherin, Snail, SLUG and α-SMA mRNA levels in MAECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and ox-LDL stimulation (100 μg / ml, 48 hours). (E) Representative images and protein quantification analysis of FKBP10, ZO-1, ZEB1, N-Cadherin, VE-Cadherin, CD31, VIM, α-SMA, Slug, Snail and TAGLN levels in HUVECs and MAECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). (F) Transwell assay showed the migration ability of HUVECs and MAECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and ox-LDL stimulation (100 μg / ml, 48 hours). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0043] Figure 4 Endothelial cell-specific FKBP10 knockdown inhibited EndMT in vivo in the example of the present invention.

[0044] (A) Representative immunofluorescence staining images of FKBP10 protein levels in the aortic root and carotid artery bifurcation segments of two groups (n = 10). Scale bar = 100 μm. (B) Representative immunofluorescence staining images of snail protein levels in the aortic root and carotid artery bifurcation segments of two groups (n = 10). Scale bar = 100 μm. (C) Representative immunofluorescence staining images of VE-Cadherin protein levels in the aortic root and carotid artery bifurcation segments of two groups (n = 10). Scale bar = 100 μm.

[0045] Figure 5In the embodiment of the present invention, FKBP10 activates the PI3K / AKT and MAPK signaling pathways by interacting with PARP1. (A) KEGG enrichment analysis was performed on siNC or siFKBP10-1, siFKBP10-2 pretreated and stimulated with ox-LDL (100 μg / ml, 48 h) to identify differentially expressed genes. (B) Representative Western blot images and protein quantification analysis of FKBP10, P-PI3K, and PI3K levels in HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). (C) Representative Western blot images of purification and size separation of input, IgG, and anti-FKBP10 on 7.5% SDS-PAGE. (D) Venn diagram showing that there are 22 proteins in the intersection between the protein profile and the reported phosphorylation-related proteins. (E-F) Co-IP experiments detected the interaction between FKBP10 and PARP1 in HUVECs treated with ox-LDL (100 μg / ml). (G) Molecular simulation and protein docking of FKBP10 and PARP1.

[0046] (H) Schematic diagram of myc-tagged full-length (WT) FKBP10 and its various deletion mutants (P1: deletion of 174-486aa, P2: deletion of 62-173aa and 286-486aa, P3: deletion of 62-285aa&399-486aa, P4: deletion of 62-398aa, EF: deletion of 62-486aa) (top). HEK 293T cells were co-transfected with His-tagged PARP1 and Myc-tagged FKBP10 or its deletion mutants or vector, whole cell lysates were evaluated by immunoprecipitation, and then immunoblotted with anti-His-tag and anti-Myc-tag. Data are expressed as mean±SD. *p<0.05, **p<0.01, ***p<0.001.

[0047] Figure 6 In the embodiment of the present invention, FKBP10 promotes the membrane localization of PARP1.

[0048] (A) Fluorescent localization and quantification of FKBP10 (green) and PARP1 (red) after pretreatment of HUVECs with ox-LDL (100 μg / ml). (B) Representative Western blot images and protein quantification of the PARP1 level in the cell membrane after treatment of HUVECs with ox-LDL (100 μg / ml, 48 h). (C) Representative Western blot images and protein quantification of the PARP1 level in the nucleus after treatment of HUVECs with ox-LDL (100 μg / ml, 48 h). (D) Fluorescent localization and quantification of PARP1 (green) and FKBP10 (red) in HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). (E) Representative Western blot images and protein quantification of the PARP1 level in the cell membrane of HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). (F) Representative Western blot images and quantification of the PARP1 level in the nucleus of HUVECs after pretreatment with siNC or siFKBP10-1, siFKBP10-2 and stimulation with ox-LDL (100 μg / ml, 48 h). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0049] Figure 7 In the example of the present invention, TAK733 binds to FKBP10, hinders the membrane translocation of PARP1, thereby inhibiting the activation of PI3K / AKT and MAPK signaling pathways.

[0050] (A) Docking prediction of FKBP10 and TAK733. (B) qRT-PCR analysis of SLUG, ZEB1, and VE-Cadherin mRNA levels in HUVECs after treatment with TAK733 (0, 2, 5 μM) and stimulation with ox-LDL (100 μg / ml, 48 h). (C) qRT-PCR analysis of SLUG, ZEB1, and VE-Cadherin mRNA levels in MAECs after treatment with TAK733 (0, 2, 5 μM) and stimulation with ox-LDL (100 μg / ml, 48 h). (D) Quantitative analysis of FKBP10, ZEB1, VIM, SLUG, and Snail levels in HUVECs after treatment with TAK733 (0, 2, 5 μM) and stimulation with ox-LDL (100 μg / ml, 48 h). (E) Transwell assay showing the migratory ability of TAK733 treatment (0, 2, 5 μM) and ox-LDL stimulation (100 μg / ml, 48 h) on HUVECs and MAECs. (F) Fluorescent localization and quantification of PARP1 (green) in HUVECs after TAK733 treatment (0, 2, 5 μM) and ox-LDL stimulation (100 μg / ml, 48 h). (G) Representative Western blot images and protein quantification analysis of PARP1 levels in the cell membrane of HUVECs after TAK733 treatment (0, 2, 5 μM) and ox-LDL stimulation (100 μg / ml, 48 h). (H) Representative Western blot images and protein quantification of nuclear PARP1 levels in HUVECs after TAK733 treatment (0, 2, 5 μM) and ox-LDL stimulation (100 μg / ml, 48 h). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed implementation manners

[0051] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0052] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0053] The present invention will be further described below in connection with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are generally in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0054] The present invention will be further explained and illustrated below through examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions specified in the following examples are generally carried out under conventional conditions.

[0055] Example

[0056] Data availability

[0057] All the research is open and transparent, and specific experimental methods and experimental materials are provided for the replication of the experiments. The FASTQ files of atherosclerotic plaques in mice with an atherosclerotic protein background (Ldlr - / - ) were obtained from the NCBI Gene Expression Omnibus (GEO) database, with the accession number GSE155513.

[0058] Patients and samples

[0059] The study has been approved by the Ethics Committee of Qilu Hospital (KYLL-202111-037-1), and all patients have given informed consent before the operation. In this study, patients over 18 years old with carotid atherosclerosis or stenosis underwent CEA surgery in the department from December 2020 to January 2023. All patients have been excluded from other causes by Doppler ultrasound, computed tomography angiography, magnetic resonance or angiography, and digital subtraction angiography (DSA).

[0060] Animals

[0061] All experimental animals used in the experiments have been approved by the Scientific Research Ethics Committee of Qilu Hospital, Shandong University (approval number: DWLL-2023-080). All mice were housed in the SPF-class animal house of Qilu Hospital, Shandong University, with specific humidity and temperature and a 12-hour light cycle. All animal experiments have passed ethical review. Male Apoe mice at 8 weeks of age - / -C57BL mice were randomly divided into two groups and were injected via the tail vein with shRNA-AAV (AAV-ICAM2-empty plasmid, control group) and Fkbp10 shRNA-AAV9 (AAV-ICAM2-shFkbp10; 5×1011 vg; Vigene Biosciences, Jinan, China), respectively. The sequence of shFkbp10 was 5′-GAAGACATGGATCTCAATAAA-3′. Four weeks after establishing the mouse model with endothelial-specific knockdown, a high-fat diet was fed for 16 weeks to establish an atherosclerosis model.

[0062] Cell culture

[0063] HUVEC and MAEC were provided by the Department of Cardiology, Qilu Hospital of Shandong University. The culture medium used for endothelial cell culture and passage was endothelial cell-specific medium (ECM) supplemented with 15% fetal bovine serum (FBS), 1% endothelial cell growth factor (P / S), and 1% penicillin-streptomycin. HEK293T cells were cultured in DMEM (CM10013, Macgene) supplemented with 10% FBS and 1% P / S. All cells were cultured at 37°C and 5% CO2. When the endothelial cell density in the six-well plate reached 50%-60%, 100 μg / ml of ox-LDL was added and the cells were treated for 48 hours.

[0064] SiRNA interference, plasmid transfection, and lentivirus infection

[0065] According to the manufacturer's protocol, short interfering RNA (siRNA) was transiently transfected into cells using Lipofectamine 3000 reagent (L3000150, Thermo Fisher Scientific). For plasmid transfection, Lipofectamine 3000 reagent (L3000150, Thermo Fisher Scientific) was also used according to the manufacturer's instructions. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze mRNA expression. The custom siRNA sequences used in this study are shown in Table 1, the shRNA sequences are shown in Table 2, and the plasmids and vectors are shown in Table 3.

[0066] Table 1. siRNA sequence information

[0067]

[0068] Table 2. shRNA sequences

[0069]

[0070] Table 3. Plasmid information

[0071]

[0072] Quantitative real-time PCR (qRT-PCR)

[0073] Total RNA extraction kit (Yishan Bio, ES-RN001). cDNA was obtained by reverse transcription using Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (11141ES60, Yeasen). Hieff qPCR SYBR Green Master Mix (No Rox) (11201ES08, Yeasen) was used as the fluorescent dye, and a Roche 480II real-time fluorescence quantitative PCR detection system (Roche; Basel, Switzerland) was used for qRT-PCR. Actin was used as the quantitative standard in all experiments, and the calculation method was ΔΔCt.

[0074] Western blot

[0075] Total protein was extracted from the cells and lysed on ice with RIPA lysis buffer containing protease inhibitor (1:50) and phosphatase inhibitor (1:50) (P0013B, Beyotime). The BCA Protein Assay Kit (P0009, Beyotime) was used to determine the protein concentration as the loading reference. A cell membrane and cytoplasm protein extraction kit (20127ES50, Yeasen) was used to separate and extract proteins from the cell membrane and cytoplasm. Cytoplasmic and nuclear protein extraction was based on a nuclear and cytoplasmic extraction kit (Pierce) (78833, Thermo Scientific). SDS-PAGE was performed using a 7.5% gel. Proteins were transferred to a PVDF membrane at a constant current of 230V for 1.5 hours, sealed with 5% non-fat milk for 1 hour, incubated overnight with the primary antibody at 4°C, and incubated with the secondary antibody, goat anti-mouse / rabbit IgG (H+L), for 1 hour. Total protein was quantified using β-actin as the reference standard, nuclear protein was quantified using H3 as the reference standard, and + Na + -K

[0076] Immunohistochemistry

[0077] Bake paraffin sections, dewax with xylene, and then repair with EDTA antigen repair solution (P0085, Beyotime), cool to room temperature, and wash three times with PBS. For frozen sections, incubate with 3% H2O2 for 10 minutes, and block with PBS containing 0.5% Triton X-100, 1% BSA, and 10% donkey serum for 30 minutes. Drop the primary antibody on the sections and incubate overnight at 4°C. Use an immunohistochemistry kit (PV-9000, ZSGB-BIO) according to the instructions. Scan the sections using the WISLEAP scanning system (WISLEAP Medical Technology Co., Ltd.). Score and count positive cells according to the staining intensity, and perform immunohistochemical analysis. The primary antibodies used are shown in Table S6.

[0078] Immunofluorescence

[0079] For immunofluorescence staining of cells, 2000 endothelial cells per well were seeded on Slide 8-well (Ibidi GmbH) overnight, and processed the next day. After 72 hours, each well was fixed with PBS solution containing 4% formaldehyde, blocked and permeabilized with 1% BSA. The cells were incubated with the primary antibody overnight at 4°C in appropriate humidity. Subsequently, the appropriate species-specific fluorescently labeled secondary antibody was diluted 1:2000 and detected at room temperature for 1 hour. TRITC Phalloidin (40734ES75, Yeasen) was used to stain F-actin. DAPI (blue) (C1002, Beyotime) was used as a nuclear stain. Images were taken using a Leica TCS SP8 confocal microscope (Leica Microsystems; Wetzlar, Germany).

[0080] For immunofluorescence staining of tissue sections, bake paraffin sections at 70°C for 30 minutes, dewax with xylene and 100% ethanol, air dry, and wash three times with PBS. Then repair with EDTA antigen repair solution (P0085, Beyotime) at 95°C for 30 minutes and cool to room temperature for 1 hour. Wash frozen sections directly three times with PBS, incubate with 3% H2O2 for 10 minutes, and block with PBS containing 0.5% Triton X-100, 1% BSA, and 10% donkey serum for 30 minutes. Incubate the primary and secondary antibodies as described above. The primary antibodies used are shown in Table S6.

[0081] HE staining

[0082] According to the American Heart Association, cross-sections were taken at 6 μm intervals from the origin of the aortic valve to the ascending aorta. Paraffin-embedded sections of the left carotid artery and frozen sections of the right carotid artery were prepared. All sections were taken from the bifurcation of the two carotid arteries. The sections were dewaxed with xylene and hydrated with ethanol of decreasing concentration. Hematoxylin staining was performed for 5 minutes, followed by washing with water, and eosin staining was performed for 3 minutes. The sections were dehydrated with ethanol of increasing concentration, cleared with xylene, and sealed with neutral gum (G8590, Solarbio). The sections were scanned using the WISLEAP scanning system (WISLEAP Medical Technology Co., Ltd.). The plaque area was viewed using NDP.view software (2.8.24).

[0083] Oil Red O staining

[0084] Remove the surface fat, fix the whole aorta with paraformaldehyde, soak it in Oil Red O dye solution for 30 minutes, and wash it with staining wash buffer. Take pictures with a surgical microscope. The frozen sections were rewarmed and dried, soaked in Oil Red O dye for 15 minutes, and washed with staining wash buffer. Seal the sections with glycerin gum. The sections were scanned using the WISLEAP scanning system (WISLEAP Medical Technology Co., Ltd.). Calculate the percentage of Oil Red O positive area using Image J (1.53C) software.

[0085] Masson staining

[0086] The frozen sections were rewarmed and dried. They were stained successively with the prepared hematoxylin staining solution, blued and washed with Masson staining solution, stained with ponceau staining solution, and stained with aniline blue staining solution. Dehydrate with absolute ethanol, clear with xylene 3 times, and seal with neutral tree gum. The sections were scanned using the WISLEAP scanning system (WISLEAP Medical Technology Co., Ltd.). Calculate the percentage of collagen positive area using Image J (1.53C) software.

[0087] Co-immunoprecipitation

[0088] The experiment was performed using Pierce TM Classic Magnetic IP / Co-IP Kit (88804, Thermo Fisher Scientific). The cell lysate was combined with the antibody (see Table S6) or IgG (3420S; Cell Signaling Technology) to bind the antigen-antibody complex to Protein A / G magnetic beads (88804, Thermo Fisher Scientific) for 1 hour. Wash twice with elution buffer and once with pure water to elute the antigen and antibody complex. Analyze it by Western blot.

[0089] Transwell assay

[0090] Add the medium containing 15% FBS to the lower layer of the 24-well plate, and add the suspended cells to the transwell chamber (3×104 cells / 300 μL). The cells passing through the 8-μm membrane were fixed with 4% paraformaldehyde and stained with crystal violet for 10 minutes. Bright-field microscope images were collected, and the cells were counted using Image J.

[0091] Protein mass spectrometry

[0092] The samples were separated by SDS-PAGE gel, and the protein bands were visualized by Coomassie Brilliant Blue staining. Proteases digested the proteins into small peptide fragments, which were sent to the company (Novogene Co., Ltd) for protein mass spectrometry analysis.

[0093] Protein-protein docking

[0094] Protein-protein docking is a computational method for predicting the near-native structure of a protein complex based on the known three-dimensional structures of two separate proteins. Docking includes sampling and scoring processes. In this study, the HDOCK server was used for protein docking. HDOCK combines physical and bioinformatics-based methods, developing an efficient molecular docking algorithm and an accurate biomolecular interaction scoring function. HDOCK samples all possible binding modes between two different proteins. Then the scoring function ranks the sampled binding modes according to their likelihood. Using the HawkDock server, the binding free energy of the two proteins was calculated by the MM / GBSA method. The binding process of the two proteins was visualized in surface representation. The binding interface of the protein-protein complex was comprehensively described and systematically analyzed using the PLIP interaction analysis platform. In addition, PyMOL was used to further supplement the interaction details.

[0095] Statistical analysis

[0096] Statistical analysis was performed using GraphPad Prism 8.3.0. Refer to the legend for the number of replicates. All data were expressed as mean values, and the error was expressed as SD. A two-tailed unpaired Student t-test was used for two groups of normally distributed data. One-way ANOVA was used to analyze multiple groups of data. For all statistical analyses performed, P < 0.05 was considered a significant difference.

[0097] Results

[0098] FKBP10 is highly expressed in endothelial cells of neovascularization in human unstable plaques

[0099] Focusing on analyzing the expression of FKBP10 in endothelial cells of mouse plaques at different time points, it was found that with long-term high-fat diet and the progression of plaques, the expression level of FKBP10 gradually increased ( Figure 1A), the endothelial cells of FKBP10+ also increased. However, at 26 weeks, compared with 18 weeks, the FKBP10-positive endothelial cells were slightly decreased. To further confirm the protein expression level and localization of FKBP0 in plaques, immunohistochemistry and immunofluorescence experiments were performed using carotid atherosclerotic plaque specimens from a previously established mouse model of high-fat diet-induced atherosclerosis. The results showed that FKBP10 was significantly upregulated in carotid plaques of the mouse model of high-fat diet-induced atherosclerosis ( Figure 1 B), and was mainly localized in CD31 + -positive endothelial cells ( Figure 1 C). To increase the reliability of the experiment, 10 stable plaques and 10 unstable plaques were randomly selected from a previously constructed human carotid atherosclerotic plaque specimen bank for immunohistochemistry. The plaque stability was evaluated according to the symptoms of the patients and the plaque stability score. The results showed that the expression level of FKBP10 was higher in unstable plaques, especially in the area of neovascularization within the plaques ( Figure 1 D). Immunofluorescence further confirmed that FKBP10 was mainly expressed in the endothelial cells of neovessels in unstable plaques, showing a high degree of consistency with the expression of CD31 ( Figure 1 E). Similarly, single-cell RNA sequencing of human carotid atherosclerotic plaques showed that FKBP10 was mainly upregulated in neovascular endothelial cells and endothelial cells undergoing EndMT ( Figure 1 F, G). So far, many studies have investigated the mechanisms by which high levels of LDL-cholesterol, especially ox-LDL, affect vascular biology and lead to the formation of atherosclerotic lesions. Therefore, HUVEC and MAEC were treated with ox-LDL (100 μg / mL, 48 h) in vitro. The results showed that ox-LDL treatment significantly increased the expression of FKBP10 in endothelial cells.

[0100] These results indicate that FKBP10 is significantly upregulated in atherosclerotic plaques and is closely related to EndMT and plaque neovascularization, which may play a key role in enhancing plaque instability.

[0101] Endothelial cell-specific knockdown of FKBP10 attenuates the atherosclerotic plaque process

[0102] Since the expression of FKBP10 is increased in atherosclerotic plaque endothelial cells, it was determined whether knockdown of FKBP10 would attenuate the progression of atherosclerotic plaques. Thus, eight-week-old ApoE - / - C57 mice were injected with Endo-Fkbp10 KD -rAAV9 via the tail vein to establish a mouse model with specific knockdown of endothelial cell FKBP10. Endo-NC was injected KD-rAAV9 was used as a control. Four weeks after injection, mice at 12 weeks of age were fed a high-fat diet for 16 weeks and sacrificed at 28 weeks of age. Aorta and carotid arteries were collected for subsequent analysis. Compared with ApoE - / - -Endo-NC KD mice, the atherosclerotic plaque areas in the aortic arch, carotid artery, and aortic root of ApoE - / - -Endo-Fkbp10 KD mice were significantly reduced ( Figure 2 B-F). In addition, Oil Red O staining also showed that the lipid lesion areas in the aortic arch, carotid artery, and aortic root of ApoE - / - -Endo-Fkbp10 KD mice were smaller than those of ApoE - / - -Endo-NC KD mice ( Figure 2 C, G, H). Masson staining showed that FKBP10 knockdown increased the collagen fiber content in atherosclerotic plaques in the aortic root and carotid artery ( Figure 2 I and J). FKBP10 knockdown also reduced macrophage infiltration. The above results indicate that during the progression of atherosclerosis, the deletion of endothelial FKBP10 is associated with a reduced atherosclerotic burden and a more favorable plaque phenotype.

[0103] FKBP10 silencing in vitro and in vivo attenuates EndMT

[0104] Based on the fact that endothelial-specific FKBP10 knockdown can inhibit the progression of atherosclerosis, the role of FKBP10 in endothelial cell function was elucidated. Endothelial FKBP10 was knocked down using siRNA, and then treated with ox-LDL (100 μg / mL) for 48 hours for transcriptome sequencing ( Figure 3 A). Gene Set Enrichment Analysis (GSEA) of differentially expressed genes revealed that FKBP10 knockdown was closely associated with the negative regulation of the EMT signaling pathway ( Figure 3 B). This result supports the finding in Figure 1 that FKBP10 is mainly highly expressed in neonatal vascular endothelial cells and endothelial cells undergoing EndMT. Since EMT refers to epithelial-to-mesenchymal transition and EndMT refers to endothelial-to-mesenchymal transition, both are characterized by increased expression of mesenchymal markers such as Snail, SLUG, and ZEB1. Based on this finding, the role of FKBP10 in EndMT was focused on. Knockdown of FKBP10 in HUVECs significantly inhibited the expression of mesenchymal markers ZEB1, Snail, N-Cadherin, VIM, SLUG, α-SMA, and TAGLN, while restoring the expression of endothelial markers ZO-1, VE-Cadherin, and CD31 under ox-LDL (100 μg / mL) stimulation.Figure 3 C). These findings indicate that FKBP10 knockdown effectively inhibits ox-LDL-induced EndMT. It has been reported that endothelial cells have significant morphological changes and exhibit enhanced migratory ability. Next, a Transwell assay was used to evaluate the effect of FKBP10 knockdown on endothelial cell migration. The results showed that FKBP10 knockdown significantly inhibited the migratory ability of HUVECs and MAECs under ox-LDL (100 μg / mL) stimulation ( Figure 3 F).

[0105] In addition, an immunofluorescence assay was performed to investigate the in vivo effect of endothelial-specific FKBP10 knockdown on EndMT. The results showed that compared with the Endo-NC KD group, the expression of FKBP10 and mesenchymal markers (Snail, N-Cadherin, VIM, SLUG, α-SMA) in plaque endothelial cells at the aortic root and carotid artery bifurcation of Endo-Fkbp10 KD group mice was inhibited. Meanwhile, the expression of the endothelial marker VE-Cadherin was partially restored ( Figure 4 A-C). Notably, TAGLN and α-SMA staining showed that the proportion of TAGLN- and α-SMA-positive areas within the plaque in the Endo-Fkbp10KD group was significantly increased. This indicates enhanced smooth muscle fiber cap components, suggesting improved plaque stability, which is closely consistent with the results shown in Figure 2 I-J.

[0106] In summary, for atherosclerotic plaques, this will significantly increase the barrier function of vascular endothelium and reduce plaque instability caused by increased endothelial cell migratory ability.

[0107] FKBP10 activates the PI3K / AKT and MAPK signaling pathways by interacting with PARP1

[0108] KEGG enrichment analysis of differentially expressed genes in RNA-seq data showed that FKBP10 knockdown mainly affected Rap1, MAPK, PI3K / AKT, and JAK / STAT signaling pathways in HUVECs ( Figure 5 A), and these signaling pathways are all closely related to protein phosphorylation. Western blot results showed that FKBP10 knockdown significantly inhibited the PI3K / AKT, ERK, JNK, and P38 signaling pathways under ox-LDL stimulation ( Figure 5 B). These findings suggest that FKBP10 may play a role in regulating intracellular phosphorylation signaling pathways. To further investigate this, immunoprecipitation and in vitro mass spectrometry were performed to screen for its target proteins ( Figure 5C). The identified proteins are listed in Table S7. By taking the intersection of the proteins that interact with FKBP10 and the proteins involved in phosphorylation activity, PARP1 was identified as a key target ( Figure 5 D). Additionally, as shown in Figure 5 E-F, in HUVECs, immunoprecipitation of FKBP10 and PARP1 respectively led to co-immunoprecipitation of PARP1 and FKBP10, but the anti-IgG antibody did not immunoprecipitate FKBP10 and PARP1. The binding domains of FKBP10 and PARP1 were further investigated by molecular simulation and protein docking. The binding free energy calculated using the MM / GBSA method by the HawkDock server was -51.45 (kcal / mol), the docking score was -258.34, and the confidence score was 0.8972. Interaction analysis was performed with PARP1 as the reference chain, revealing detailed binding characteristics, where purple represents FKBP10 and yellow represents PARP1. A total of 29 hydrogen bonds (within ) and 2 salt bridges were identified. As shown in Figure 5 G(a), a hydrogen bond (blue solid line) was formed between LYS-579 of PARP1 and ARG-13 of FKBP10. In Figure 5 G(b), hydrogen bonds were formed between LYS-607 of PARP1 and TYR-83 of FKBP10, GLU-576 / GLU-547 of PARP1 and ASN-86 of FKBP10, GLY-781 of PARP1 and LYS-240 of FKBP10, GLY-549 of PARP1 and HIS-68 / THR-189 of FKBP10, LYS-703 of PARP1 and ASP-644 of FKBP10. In addition, TYR-645 of PARP1 interacted with THR-244 and GLY-243 of FKBP10 through hydrogen bonds. Figure 5 G(c) shows that hydrogen bonds were formed between ASN-630 of PARP1 and ASN-162 of FKBP10, and between LYS-940 of PARP1 and PRO-264 of FKBP10. Other interactions include hydrogen bonds between ASP-202 of FKBP10 and GLU-883 of PARP1, and between TYR-201 of FKBP10 and GLN-707 / GLU-642 of PARP1. In addition, a salt bridge (yellow dashed line) was formed between ARG-704 of PARP1 and ASP-192 of FKBP10, along with a hydrogen bond formed by TYR-2237 of FKBP10. A hydrogen bond was also formed between LYS-551 of PARP1 and SER-190 of FKBP10. Figure 5G(d) highlights the hydrogen bonds between LYS-893 of PARP1 and GLU351 of FKBP10, between SER-939 of PARP1 and HIS-346 of FKBP10, between GLU-351 of FKBP10 and ASN-352 of PARP1, between VAL-886 / GLN-717 of PARP1 and ASN-352 of FKBP10, between GLN-178 of PARP1 and ILE-358 of FKBP10, between GLN-728 of PARP1 and GLY-355 of FKBP10, and between GLN-722 of PARP1 and ASP-356 of FKBP10. Additionally, a large number of hydrophobic interactions were also observed (gray dotted lines), further stabilizing the FKBP10-PARP1 complex. Based on the molecular docking results and the functional domain of FKBP10, five truncated mutant plasmids of FKBP10 were designed, each loaded with a Myc-Tag( Figure 5 H, upper), and Co-IP experiments were performed to confirm their interaction. The results showed that only WT and P2 could maintain a close interaction with PARP1, while other mutant types could not. This indicates that the PPIase 2 domain plays a key role in the interaction between FKBP10 and PARP1.( Figure 5 H, lower). However, when FKBP10 was knocked out, it did not affect the protein expression level of PARP1.

[0109] FKBP10 promotes the membrane localization of PARP1

[0110] To further prove this, immunofluorescence was used to detect the subcellular co-localization of FKBP10 and PARP1. The results showed that FKBP10 and PARP1 were mainly co-localized in the cytoplasm( Figure 6 A). Interestingly, PARP1 was distributed in the nucleus, cytoplasm, and cell membrane. However, under ox-LDL stimulation, the membrane localization of PARP1 increased significantly( Figure 6 A). Proteins from the cell membrane and nucleus were extracted, and Western blot was used to prove this phenomenon( Figure 6 B-C).

[0111] To prove whether this phenomenon was related to FKBP10, FKBP10 in HUVEC was subsequently knocked down and treated with ox-LDL for 48 hours. Immunofluorescence results showed that FKBP10 knockdown reduced the membrane localization of PARP1 and increased its nuclear localization( Figure 6 D). This conclusion was further verified by Western blot( Figure 6(E-F), similar results were also shown in MAEC. This result indicates that the interaction between FKBP10 and PARP1 inhibits the nuclear translocation of PARP1, promotes its migration to the cell membrane, and activates the PI3K / AKT, ERK, JNK, and P38 signaling pathways.

[0112] TAK733 binds to FKBP10 and inhibits the nuclear translocation of FKBP10, thereby inhibiting the activation of the PI3K / AKT and MAPK signaling pathways

[0113] According to the above mechanism, small molecule drugs that can be used to treat atherosclerosis were identified. Using the differentially expressed genes of RNA-seq and cMap for small molecule drug screening, TAK733 and Iniparib were initially identified as candidate drugs. Molecular docking analysis showed that TAK733 binds to FKBP10 with an affinity score of -6.8 kcal / mol. TAK733 forms hydrogen bonds (blue solid lines) with ASN-367, SER-296, GLY-300, and ASP-299 of FKBP10, a halogen bond (cyan solid line) with PRO-164, and hydrophobic interactions (gray dashed lines) with PRO-164 ( Figure 7 A). Under ox-LDL stimulation, the addition of TAK733 (2 μM, 5 μM) significantly inhibited EndMT in HUVEC and MAEC ( Figure 7 B-D), while Iniparib had no such effect. The Transwell experiment further showed that TAK733 significantly reduced the migration ability of HUVEC and MAEC and significantly changed the morphology of endothelial cells ( Figure 7 E). To further explore the mechanism by which TAK733 inhibits EndMT, it was found that TAK733 significantly inhibited the membrane translocation of PARP1 induced by ox-LDL ( Figure 7 F), which was consistent with the effect observed with FKBP10 knockdown. Similarly, TAK733 inhibited the activation of the PI3K / AKT, ERK, JNK, and P38 signaling pathways. Therefore, TAK733 has great potential as a candidate drug for stabilizing atherosclerotic plaques by inhibiting EndMT.

[0114] Matters not covered by this invention are well-known technologies.

[0115] The above embodiments are only used to illustrate the technical concept and features of the present invention, and their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of FKBP10 in the preparation of an atherosclerotic detection product; the atherosclerotic detection product is used for screening, (auxiliary) diagnosis, monitoring or prognostic evaluation of atherosclerosis.

2. The application according to claim 1, wherein The substance for detecting the expression level of FKBP10 includes reagents for detecting the expression level of the FKBP10-encoding gene based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing, and / or reagents for detecting the protein (enzyme) expression level of FKBP10 based on immunoassay methods; The product includes primers, probes, (gene or protein) chips, detection kits and detection devices.

3. A system for detecting atherosclerosis, characterized in that, The system includes: An acquisition module configured to: acquire the expression level of FKBP10 in a sample to be tested of a subject; An analysis module configured to: analyze and judge the disease condition of the subject according to the expression level of FKBP10 of the subject obtained by the acquisition module.

4. The system according to claim 3, characterized in that The sample to be tested can be an atherosclerotic plaque sample or an endothelial cell sample of the subject, and further an endothelial cell sample of the neovascularization in the atherosclerotic plaque.

5. The system according to claim 3, wherein The detection of atherosclerosis is for screening, (auxiliary) diagnosis, monitoring or prognostic evaluation of atherosclerosis, and further includes the detection and analysis of endothelial-mesenchymal transition and plaque neovascularization in atherosclerosis.

6. Use of FKBP10 as a target in the preparation and / or screening of atherosclerotic drugs.

7. The application according to claim 6, characterized in that, The method for screening atherosclerotic drugs includes: 1) Treating a system expressing and / or containing FKBP10 with a candidate substance; setting a control without treating with the candidate substance; 2) After completing step 1), detecting the expression level of FKBP10 in the system; compared with the control, if the expression level of FKBP10 in the system treated with the candidate substance is significantly down-regulated, the candidate substance can be used as a candidate drug for preventing and / or treating atherosclerosis.

8. The application according to claim 7, characterized in that The system is a solution system, a cell system (such as endothelial cells), a tissue system, an organ system or an animal system.

9. Use of a substance that inhibits the expression of FKBP10 or reduces its activity in any one or more of the following: (a) Inhibiting the membrane translocation of PARP1 or preparing a product for inhibiting the membrane translocation of PARP1; (b) Inhibiting the activation of the PI3K / AKT and MAPK signaling pathways or preparing a product for inhibiting the activation of the PI3K / AKT and MAPK signaling pathways; (c) Inhibiting endothelial-mesenchymal transition or preparing a product for inhibiting endothelial-mesenchymal transition; (d) A product for preventing and / or treating atherosclerosis.

10. The application according to claim 9, characterized in that, The substance that inhibits the expression of FKBP10 or reduces its activity includes RNA interference molecules or antisense oligonucleotides against FKBP10, small molecule inhibitors (including TAK733), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout; The product is a drug or an experimental reagent for non-medical use.

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