Biomarker for coronary atherosclerosis bifurcation lesion and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a biological marker for coronary atherosclerosis bifurcation lesion and application of the biological marker. The invention discloses application of PlexinD1 as a marker in preparation of a product for treating, preventing or diagnosing bifurcation lesion. The product comprises a reagent or a kit. Compared with imaging examination means such as coronary CTA, coronary angiography, OCT, IVUS, molecular imaging and the like, the bifurcation lesion can be recognized more economically, conveniently and quickly, no special requirement for diagnostic equipment and reagents is needed, and batch measurement of samples can be achieved only through a single ELISA kit.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a biomarker for coronary artery atherosclerotic bifurcation lesions and its applications. Background Art

[0002] Coronary atherosclerotic heart disease, abbreviated as coronary heart disease, is the disease with the highest lethality and disability rate globally. The rupture and erosion of atherosclerotic vulnerable plaques are the main causes of adverse cardiovascular events. Numerous studies have confirmed that atherosclerotic vulnerable plaques are more distributed in the bifurcation parts of coronary arteries, called bifurcation lesions. The blood revascularization strategy for bifurcation lesions has always been a difficult point and the focus of debate in clinical treatment. Analyzing the occurrence and development mechanism of bifurcation lesions and early detecting bifurcation lesions are of great significance for formulating intervention strategies, implementing precise interventions, and reducing the occurrence of cardiovascular events.

[0003] In recent years, researchers from the European, American, and Asian Society of Cardiovascular Interventional Cardiology have, based on coronary angiography images, defined bifurcation lesions as coronary artery stenoses adjacent to and / or located at the side branches with a diameter ≥ 2 mm and a stenosis rate greater than 50% and a minimum diameter distance from the bifurcation point POB ≤ 4 mm, and proposed Medina classification, Lefevre classification, and Duke classification according to the positions of bifurcation lesions. Currently, there are many creative technologies and methods for interventional treatment of bifurcation lesions, but the diagnosis and classification of bifurcation lesions still mainly rely on imaging examination means such as coronary angiography, coronary CTA, OCT, IVUS, and molecular imaging. Coronary CTA requires intravenous injection of iodine contrast agent, has radiation, and is not applicable to people with iodine allergy. Coronary angiography, OCT, and IVUS are all invasive interventional operations, with complex procedures, expensive examinations, and radiation exposure. Although the molecular imaging means targeting specific molecular targets of bifurcation lesions is a non-invasive operation, it has high requirements for imaging probes and imaging equipment and has not been widely applied clinically. Therefore, there is an urgent need for a technical solution to address the above deficiencies of the existing technologies, such as specific peripheral serum biomarkers for bifurcation lesions, but there is no relevant research currently.

[0004] As an explanation for the occurrence and development mechanism of bifurcation lesions, the hemodynamic hypothesis has currently received extensive attention and recognition. This hypothesis holds that the oscillatory shear stress formed by blood flow at the bifurcation part of blood vessels promotes the occurrence and development of atherosclerosis compared with the laminar shear stress at the straight part of blood vessels. Research shows that vascular endothelial cells have a variety of molecular sensors that can receive the turbulent mechanical signals at the bifurcation part and convert them into biological signals. While causing changes in their own functions, the signals are further transmitted to cells such as smooth muscle cells and macrophages, ultimately jointly promoting the occurrence and development of atherosclerosis at the bifurcation part. Thus, it can be seen that the occurrence and development mechanism of bifurcation lesions may become an important entry point for exploring peripheral serum biomarkers and improving the existing technical solutions. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a biomarker for coronary atherosclerotic bifurcation lesions and its application based on the ELISA detection method. The present invention provides a biomarker capable of identifying coronary atherosclerotic bifurcation lesions - peripheral serum protein PlexinD1, which can preferably distinguish between patients with and without bifurcation lesions among patients with acute coronary syndrome, and provides a non-invasive diagnostic method for bifurcation lesions.

[0006] To achieve the above object, the present application provides the following technical solutions: The application of PlexinD1 as a biomarker in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions, wherein the products include reagents or reagent kits.

[0007] Preferably, the application of peripheral serum PlexinD1 as a biomarker in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions.

[0008] The application of a drug that inhibits the expression of PlexinD1 in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions.

[0009] The application of PlexinD1 and M1 macrophages as biomarkers in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions, wherein the products include reagents or reagent kits.

[0010] Preferably, the application of peripheral serum PlexinD1 and M1 macrophages as biomarkers in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions.

[0011] The application of a drug that inhibits the expression of PlexinD1 and the polarization of M1 macrophages in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions.

[0012] The application of PlexinD1, M1 macrophages and PTGS2 as biomarkers in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions, wherein the products include reagents or reagent kits.

[0013] The application of a drug that inhibits the expression of PlexinD1 and the polarization of M1 macrophages and enhances the activity of PTGS2 in the preparation of products for the treatment, prevention or diagnosis of bifurcation lesions.

[0014] Beneficial Effects: Compared with imaging examination methods such as coronary CTA, coronary angiography, OCT, IVUS, and molecular imaging, the present invention can more economically, conveniently and quickly identify bifurcation lesions, and has no special requirements for diagnostic equipment and reagents, and only a single ELISA kit is required to achieve batch determination of samples. Brief Description of the Drawings

[0015] The accompanying drawings of the specification, which form a part of this application, are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation of this application. Among them: Figure 1 Immunofluorescence staining of PlexinD1 (green) and iNOS (red) in carotid artery bifurcation sections of ApoE- / - mice fed a high-fat diet for 10 weeks and 20 weeks; Figure 2 Schematic diagram of an in vitro model of endothelial cell-macrophage co-culture under shear stress (LS or OS) intervention. Among them, A is a peristaltic pump, B is a direction changer, C is a culture medium bottle, D and E are smoothing devices, F is a parallel plate flow chamber, and G is a transwell chamber; Figure 3 The number of differentially expressed proteins in the proteomics detection results of endothelial cells co-cultured with macrophages (n = 3) under oscillatory shear stress and laminar shear stress in the in vitro model; Figure 4 Heat map of cluster analysis of the proteomics detection results of endothelial cells co-cultured with macrophages (n = 3) under oscillatory shear stress and laminar shear stress in the in vitro model; Figure 5 Protein-protein interaction network analysis (PPI) of the proteomics detection results of endothelial cells co-cultured with macrophages (n = 3) under oscillatory shear stress and laminar shear stress in the in vitro model; Figure 6 Western blot results of the response of endothelial cell PTGS2 to oscillatory shear stress mediating the upregulation of PlexinD1 and M1 polarization of macrophages in the in vitro model; Figure 7 Schematic diagram of the quantitative coronary angiography (QCA) analysis and measurement of the bifurcation lesion in the patient's angiographic image; Figure 8 Difference in PlexinD1 levels in peripheral blood between the bifurcation lesion group and the non-bifurcation lesion group of ACS patients; Figure 9 ROC curve of peripheral blood PlexinD1 for diagnosing bifurcation lesions in ACS patients. Detailed implementation manners

[0016] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments. Each example is provided by way of explanation of the present application rather than a limitation thereof. In fact, those skilled in the art will appreciate that modifications and variations can be made to the present application without departing from the scope or spirit thereof. For example, features shown or described as part of one embodiment can be used in another embodiment to yield yet another embodiment. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the embodiments of the present invention shall fall within the scope of protection of the embodiments of the present invention.

[0017] I. Screening of bifurcation lesion characteristic markers 1. Materials and methods In an animal model, ApoE - / - mice were selected. After being fed with normal feed until 6 - 7 weeks of age, they were switched to a high - fat diet (Spearf, D12079B) and continued to be fed for 10 weeks and 20 weeks to construct an atherosclerosis model.

[0018] 1.1 Carotid artery sampling ① Make an abdominal incision with a transverse incision in the upper part of the small intestine area of the mouse. Extend the incision along the outer side of the abdominal wall towards the cephalic end until reaching the lower edge of the lateral rib, exposing the middle and upper structures in the abdominal cavity; Cut through the diaphragm to allow the lungs to retract, then expand the tear on both sides and approach it to the abdominal wall incision; Cut both sides of the ribs and continue to extend the incision towards the cephalic end until the atrium (or the thymus covering the atrium) is exposed; ② Clamp the xiphoid process, turn the entire thoracic cage outward and fix it towards the cephalic end to fully expose the heart. Puncture the left ventricle with an injection needle, keep the needle in the left ventricle, gently push the syringe to confirm that the liquid does not leak, and then clamp the heart wall and the needle together with a hemostat to prevent the needle from moving; ③ Cut through the right auricle to bleed, and start slowly injecting 20 mL of perfusion saline solution until the liquid flowing out of the right auricle is basically colorless. The perfusion saline solution formula is 500 mL of PBS + 800 uL of heparin, and the injection speed is controlled at 0.15 - 0.2 mL / s; ④ Carefully remove the saline syringe, replace it with a syringe filled with 20 mL of fixative (invert and gently flick the syringe to remove air bubbles at the interface), continue to inject the fixative, and after the mouse stops twitching, halve the perfusion speed until the perfusion is completed; ⑤ Remove the perfusion instrument, transfer the mouse carcass to a clean tray, and separate the carotid artery and aorta of the mouse under a stereomicroscope. Pay attention to gentle movements. The removed blood vessels are rinsed in a small beaker filled with fixative; ⑥ Put all the specimens into a 1.5 mL centrifuge tube filled with neutral buffered formaldehyde fixative (the specimens should not pile up more than 1 / 3 of the liquid level depth), and make good marks; ⑦ Transfer to a refrigerator at 4 - 8 °C and continue to soak and fix.

[0019] (2)Paraffin-embedded sections of the carotid artery bifurcation ① Place the trimmed tissue and corresponding labels into a dehydration cassette, put the cassette into a hanging basket, and dehydrate it successively with different concentrations of alcohol in the following order: 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min, alcohol-benzene for 5 - 10 min, xylene I for 5 - 10 min, xylene II for 5 - 10 min, wax I for 1 h, wax II for 1 h, wax III for 1 h.

[0020] ② Embed the dehydrated tissue in an embedding machine. First, put the melted wax into an embedding frame. Before the wax solidifies, take out the tissue from the dehydration cassette, place it into the embedding frame according to the requirements of the embedding surface, and attach the corresponding label. Place the embedding frame on a -20 °C freezing table to cool. After the wax solidifies, take out the wax block and trim its shape.

[0021] ③ Place the trimmed wax block on a microtome and section at the carotid artery bifurcation site, with each section being 4 μm thick. Float the sections on warm water at 40 °C, flatten the tissue, then pick up the tissue with a glass slide and dry it in an oven at 60 °C. After the water evaporates and the wax block melts, take it out and store it at room temperature for later use.

[0022] (3)Immunofluorescence staining of sections of the carotid artery bifurcation ① Deparaffinize the sections: xylene I for 20 min, xylene II for 10 min, xylene III for 10 min; ② Rehydrate successively with different concentration gradients of alcohol in the following order: 100% alcohol for 10 min, 100% alcohol for 10 min, 95% alcohol for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, distilled water for 3 min, distilled water for 3 min, PBS wash for 5 min; ③ Antigen retrieval: Place the sections in a 0.1 mol / L citrate buffer solution with pH = 6.0, use a microwave oven at 100 power for 5 min until slightly boiling, then use 50 power for 10 min. After stopping heating, let it cool naturally for 20 - 30 min; ④ Wash with PBS for 3 min, wipe off the PBS outside the specimen with filter paper, and add 3% hydrogen peroxide to eliminate endogenous peroxidase; ⑤ Wash with PBS for 3 min × 5 times, wipe off the PBS outside the specimen with filter paper, add blocking serum, and block at 37 °C in a wet box for 1 h; ⑥ Wipe off the blocking solution with filter paper, add primary antibodies against Plexin D1 and iNOS at appropriate concentrations, incubate overnight at 4 °C in a humidified box, wash with PBS for 3 min × 5 times to remove the primary antibodies, and wipe off the PBS outside the specimen with filter paper; ⑦ Add the fluorescent secondary antibody, incubate in the dark at room temperature in a humidified box for 1 h, wash with PBS for 3 min × 5 times to remove the secondary antibody, and wipe off the PBS outside the specimen with filter paper; ⑧ Add the anti-fluorescence quenching mounting medium containing DAPI, cover with a coverslip, seal with nail polish, observe under a fluorescence microscope, and scan with a slide scanner.

[0023] Construct an endothelial-macrophage co-culture model under different shear stress interventions ( Figure 2 ). Attach endothelial cells and macrophages to the upper and lower sides of the transwell chamber membrane, install it in a flat plate flow chamber, and add oxLDL to the chamber to simulate the lipid-oxidative stress microenvironment in plaques. After filling the device with the culture medium, turn on the peristaltic pump to provide power for the overall circulation system and set the required rotation speed. When in laminar flow, the direction changer keeps the two switches on the left closed and the two switches on the right open, so that the liquid in the flat plate flow chamber flows stably in one direction to apply a shear stress of 0 + 12 dyn / cm 2 to the endothelial cells; when in oscillatory shear stress, the switches on both sides of the direction changer open and close alternately, so that the liquid in the flat plate flow chamber oscillates back and forth to apply a shear stress of 0 ± 4 dyn / cm 2 to the endothelial cells. After 24 h of intervention, take out the transwell chamber, wash with PBS, scrape off the endothelial cells with a cell scraper, quickly freeze them in liquid nitrogen, and store them in a -80 °C refrigerator. Collect 3 groups of samples under each shear stress.

[0024] In an in vitro model, perform proteomics detection on endothelial cells co-cultured with macrophages under oscillatory shear stress and laminar shear stress (n = 3), and the steps are as follows: 1.2 Protein extraction and digestion All samples were prepared by in-solution digestion method.

[0025] Add the detergent (final concentration: 20 mM) to each group of endothelial cell samples above, and incubate at 37 °C for 2 h. After cooling to room temperature, add an alkylating agent with a final concentration of 40 mM, mix at 600 rpm for 1 min, and then incubate in the dark for 30 min. Sequentially add reagent C (3 μL) and reagent D (5 μL), heat to 95 °C to terminate the reaction, and then digest with trypsin at 37 °C for 2 h. Finally, desalt the peptides of each sample, concentrate by vacuum centrifugation, and redissolve in 0.1% (v / v) formic acid. Estimate the peptide content by ultraviolet spectral density at a wavelength of 280 nm. For DIA experiments, add iRT (index retention time) calibration peptides to the standard samples (400 ng / sample).

[0026] 1.3 Mass spectrometry analysis for data-independent acquisition (DIA) Perform DIA analysis on a timsTOF Pro mass spectrometer (Bruker), which is coupled with nanoflow eluent (Bruker) liquid chromatography in data-independent acquisition (DIA) mode for 60 min. The mass spectrometer operates in positive ion mode and collects ion mobility MS spectra of ions within the m / z range defined by a single 100 ms TIMS scan of a single 100 ms TIMS scan according to the m / z ion mobility plane. During PASEF MSMS scans, the collision energy increases linearly with mobility, from 20 eV at 1 / K0 = 0.60 Vs / cm 2 to 59 eV at 1 / K0 = 1.60 Vs / cm 2

[0027] 1.4 Mass spectrometry data analysis Analyze DIA data using SpectronautTM 14.4.200727.47784 to search the database. The main software parameter settings are as follows: the retention time prediction type is dynamic iRT, interference correction at the MS2 level is enabled, and cross-run normalization is enabled. All results are filtered with a cut-off value of Q = 0.01 (equivalent to FDR < 1%).

[0028] Bioinformatics analysis 2.1 Cluster analysis ​Cluster 3.0 (http: / / bonsai.hgc.jp / mdehoon / software / cluster / software.htm) and Java Treeview software (http: / / jtreeview.sourceforge.net) were used to perform hierarchical clustering analysis. When performing hierarchical clustering, the Euclidean distance algorithm for similarity measurement and the average linkage clustering algorithm for clustering (clustering using the centroid of the observations) were selected. In addition to the dendrogram, heatmaps are usually presented as a visual aid.

[0029] 2.2 Protein-protein interaction analysis Protein-protein interaction (PPI) information of the proteins under study was retrieved from the IntAct molecular interaction database (http: / / www.ebi.ac.uk / intact / ) by their gene symbols or using the STRING software (http: / / string-db.org / ). The results were downloaded in XGMML format and imported into Cytoscape software (http: / / www.cytoscape.org / , version 3.2.1) to visualize and further analyze the functional protein-protein interaction network. In addition, the degree of each protein was calculated to evaluate the importance of the protein in the PPI network.

[0030] Differential protein PTGS2 with Fold change > 2 or < 0.5, P < 0.05 and located at the center of the protein-protein interaction network PPI was identified. Western blot verification in vitro found that overexpression of PTGS2 in endothelial cells could reduce the upregulation of PlexinD1 and M1 polarization markers in co-cultured macrophages induced by oscillatory shear stress, that is, endothelial cell PTGS2 responded to oscillatory shear stress by downregulation, inducing M1 macrophage polarization mediated by downstream macrophage PlexinD1.

[0031] II. Determination of peripheral serum markers for bifurcation lesions Bifurcation lesions were defined as coronary artery stenoses occurring adjacent to and / or at clinically significant side branches. On coronary angiography, the diagnostic criteria for bifurcation lesions were: coronary artery stenoses adjacent to and / or at side branches with a diameter ≥ 2 mm with a stenosis rate greater than 50% and a minimum diameter distance from the bifurcation point POB ≤ 4 mm. 72 patients with acute coronary syndrome admitted to the Fourth and Sixth Medical Centers of the Chinese PLA General Hospital from October 2022 to June 2023 were divided into a bifurcation lesion group and a non-bifurcation lesion group according to coronary angiography images. Peripheral serum of the patients was collected and PlexinD1 protein was detected using an ELISA kit as follows: (1)Sample addition: Standard wells and test sample wells are set up respectively. Add 100 μL of standard product or test sample, taking care not to have air bubbles. Add the sample to the bottom of the ELISA plate wells, try not to touch the well walls, gently shake to mix evenly, cover the ELISA plate with a lid or film, and incubate at 37 °C for 2 h. To ensure the validity of the experimental results, use a new standard product solution for each experiment.

[0032] (2)After incubating for 2 h, discard the liquid in the wells, shake dry, wash the plate 3 times, add 250 μL of washing solution (1x) to each well, soak for 1 - 2 min each time, and shake dry.

[0033] (3)Add 100 μL of biotinylated conjugate (1x) to each well (prepared by mixing 1 μL of biotinylated conjugate with 99 μL of biotinylated conjugate diluent, gently mix evenly, and prepare within 1 h before use), and incubate at 37 °C for 1 h.

[0034] (4)After incubating for 1 h, discard the liquid in the wells, shake dry, wash the plate 3 times, add 250 μL of washing solution (1x) to each well, soak for 1 - 2 min each time, and shake dry.

[0035] (5)Add 100 μL of enzyme - labeled avidin (1x) to each well (prepared by mixing 1 μL of enzyme - labeled avidin with 99 μL of enzyme - labeled avidin diluent, gently mix evenly, and prepare within 1 h before use), and incubate at 37 °C for 1 h.

[0036] (6)After incubating for 1 h, discard the liquid in the wells, shake dry, wash the plate 5 times, add 250 μL of washing solution (1x) to each well, soak for 1 - 2 min each time, and shake dry.

[0037] (7)Sequentially add 100 μL of TMB chromogenic reagent to each well, and develop color at 37 °C in the dark for 15 - 20 min (within 20 min, at this time, the first 3 - 4 wells of the standard product are visibly blue - gradient, and the gradient of the last 3 - 4 wells is not obvious, then terminate).

[0038] (8)Sequentially add 50 μL of stop solution to each well to terminate the reaction (at this time, the blue immediately turns yellow). The addition order of the stop solution should be as consistent as possible with the addition order of the substrate solution. To ensure the accuracy of the experimental results, add the stop solution as soon as possible after the substrate reaction time has elapsed.

[0039] (9)Measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader. Detect within 5 min after adding the stop solution.

[0040] (10)Result calculation: A standard curve was made using the professional software "Curve Exert 1.4". Taking the OD value of the standard as the abscissa and the concentration of the standard as the ordinate, a standard curve was plotted on a graphing software. The regression equation of the standard curve with the highest r value and the best fitting degree was selected. The OD value of the sample was substituted into the equation to calculate the sample concentration, and then multiplied by the dilution factor. Or, taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), a standard curve was plotted on semi-logarithmic graph paper. According to the OD value of the sample, the corresponding concentration was found from the standard curve and then multiplied by the dilution factor to obtain the actual concentration of the sample.

[0041] III. Results and Analysis Currently, multiple studies have shown that M1 macrophages can increase plaque instability and promote plaque rupture through various ways such as secreting inflammatory factors, secreting extracellular matrix metalloproteinase MMP, mediating microcalcification deposition, and apoptosis necrosis, thus leading to the occurrence of cardiovascular diseases.

[0042] In this application, section staining showed that in atherosclerotic plaques at the bifurcation of the carotid artery in mice, PlexinD1 co-localized with M1 macrophage markers and its expression increased with the progression of the lesion (as Figure 1 shown). WB analysis of the in vitro shear stress intervention model ( Figure 2 ) showed that PlexinD mediated the polarization of M1 macrophages induced by oscillatory shear stress ( Figure 6 ). Proteomic analysis of endothelial cells co-cultured with macrophages under OS and LS interventions in the in vitro cell model found 80 up-regulated proteins and 66 down-regulated proteins ( Figure 3 ), and a heat map was plotted ( Figure 4 ), and the endothelial cell differential protein PTGS2 located at the key node of the protein interaction network was found ( Figure 5 ). WB analysis under the in vitro shear stress intervention model confirmed that endothelial cell PTGS2 could respond to the intervention of oscillatory shear stress and reduce the downstream PlexinD1-mediated macrophage M1 polarization ( Figure 6 ), further improving the molecular evidence chain of oscillatory shear stress causing PlexinD1-mediated M1 macrophage polarization through endothelial cell PTGS2. Thus, it can be seen that PlexinD1 is closely related to the occurrence and development of bifurcation lesions and can be used as one of the potential markers for bifurcation lesions.

[0043] Regarding the role of PlexinD1 as a peripheral serum biomarker, previous studies have measured the levels of peripheral serum PlexinD1 in people with different degrees of obesity, but no differences have been found. Currently, there is no study on the difference in the level of peripheral blood PlexinD1 and its diagnostic value for bifurcation lesions. In this regard, the results of this technical solution show that the level of peripheral serum PlexinD1 in the bifurcation lesion group diagnosed by coronary angiography is significantly higher than that in the non-bifurcation lesion group (P < 0.05) ( Figure 7 and Figure 8 ). Figure 9 The ROC curve in

[0044] shows that the AUC is 0.6532; when 167.4 pg / ml is selected as the cut-off value, the diagnostic sensitivity of the peripheral serum PlexinD1 level for bifurcation lesions is 0.58, and the specificity is 0.79. In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0045] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of PlexinD1 as a biomarker in the preparation of products for treating, preventing or diagnosing bifurcation lesions, said products comprising reagents or kits.

2. Use of Plexin D1 as a marker according to claim 1 in the preparation of products for treating, preventing or diagnosing bifurcation lesions, characterized in that, Use of peripheral serum PlexinD1 as a biomarker in the preparation of products for treating, preventing or diagnosing bifurcation lesions.

3. Use of a drug that inhibits PlexinD1 expression in the preparation of products for treating, preventing or diagnosing bifurcation lesions.

4. Use of PlexinD1 and M1 macrophages as biomarkers in the preparation of products for treating, preventing or diagnosing bifurcation lesions, said products comprising reagents or kits.

5. Use of Plexin D1 and M1 macrophages as markers according to claim 1 in the preparation of products for treating, preventing or diagnosing bifurcation lesions, characterized in that, Use of peripheral serum PlexinD1 and M1 macrophages as biomarkers in the preparation of products for treating, preventing or diagnosing bifurcation lesions.

6. Use of a drug that inhibits PlexinD1 expression and M1 macrophage polarization in the preparation of products for treating, preventing or diagnosing bifurcation lesions.

7. Use of PlexinD1, M1 macrophages and PTGS2 as biomarkers in the preparation of products for treating, preventing or diagnosing bifurcation lesions, said products comprising reagents or kits.

8. Use of a drug that inhibits PlexinD1 expression and M1 macrophage polarization and enhances PTGS2 activity in the preparation of products for treating, preventing or diagnosing bifurcation lesions.

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