A biomarker ADAMTS8 for detecting acute aortic dissection and its application

Abstract

The present invention discloses a biomarker ADAMTS8 for detecting acute aortic dissection and its application, and relates to the field of biomedicine technology. The present invention has great clinical value and practical significance for the early diagnosis of acute aortic dissection, long-term prognosis and improvement of quality of life by screening serum markers for early diagnosis of patients with aortic dissection. The present invention obtained the differentially expressed gene ADAMTS8 of aortic dissection through spatial transcriptomics screening, and used it as a molecular marker with greater sensitivity and specificity. It has been verified by ELISA, proving that ADAMTS8 may be involved in the pathogenesis of acute aortic dissection, and can be used as an early diagnostic biomarker and a new therapeutic target for acute aortic dissection, which provides a new direction for the clinical early diagnosis and long-term prognosis evaluation of aortic dissection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a biomarker ADAMTS8 for detecting acute aortic dissection and its application. Background Art

[0002] Aortic dissection, with its insidious onset and high mortality rate, has become a critical cardiovascular disease that poses a serious threat to human health. As a critical cardiovascular emergency, aortic dissection lacks an effective early warning system. Aortic dissection typically presents with no obvious symptoms, and patients often present with sudden chest and abdominal pain. Without prompt diagnosis and treatment, the early mortality rate is extremely high. Major causes of death include cardiac complications such as aortic rupture, aortic regurgitation, acute cardiac tamponade, acute myocardial infarction, and acute heart failure, as well as complications of poor organ perfusion. While innovative advances in minimally invasive endovascular repair techniques, the development of new aortic interventional devices, and continuous improvements in surgical procedures have significantly improved the diagnosis and treatment rates of aortic dissection, the surgical complexity remains significant, and the mortality rate remains high. Therefore, early diagnosis and prevention of aortic dissection are crucial. Currently, the diagnosis of aortic dissection relies primarily on medical history, electrocardiograms (ECGs), chest X-rays, aortic angiography, transesophageal ultrasound, and aortic computed tomography. Aortic angiography and aortic computed tomography (CT) have a sensitivity of nearly 100% for diagnosing aortic dissection. However, these imaging tests are time-consuming and, in most patients, performed only when the disease is suspected, often fail to effectively alert patients to the possibility of aortic dissection. Therefore, identifying biomarkers for early diagnosis and prevention of aortic dissection is crucial.

[0003] In recent years, research on peripheral blood biomarkers for aortic dissection has primarily focused on macromolecular proteins such as smooth muscle myosin heavy chain, soluble elastin fragments, D-dimer, soluble oncogenic inhibitor 2, and matrix metalloproteinases, as well as a subset of small molecule markers, including microRNAs and small metabolites. Smooth muscle myosin heavy chain and soluble elastin fragments are elevated early in the course of the disease, limiting their clinical application and preventing their routine use. D-dimer is widely used, but its low plasma D-dimer levels in younger patients, those with shorter tear lengths, and those with pseudoembolism may lead to misdiagnosis, limiting its clinical application. Therefore, the search for biomarkers with high sensitivity and specificity, ease of use, safety, and cost-effectiveness is of great practical significance for disease prevention and diagnosis. Summary of the Invention

[0004] The purpose of the present invention is to provide a biomarker ADAMTS8 for detecting acute aortic dissection and its application to solve the problems existing in the above-mentioned prior art. ADAMTS8 screened by the present invention can be used as a diagnostic biomarker for acute aortic dissection, providing a new direction for the clinical early diagnosis and long-term prognosis evaluation of acute aortic dissection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a biomarker for detecting acute aortic dissection. The biomarker is a disintegrin metallopeptidase 8 containing a type 1 platelet tuberculin motif.

[0007] The present invention also provides use of a reagent for detecting the expression level of a disintegrin metallopeptidase 8 containing a platelet tuberculin type 1 motif in plasma in the preparation of a diagnostic product for acute aortic dissection.

[0008] Furthermore, the diagnostic product is a kit, a reagent or a chip.

[0009] Furthermore, the reagent is an ELISA detection reagent.

[0010] The present invention also provides a diagnostic product for acute aortic dissection, comprising a reagent for detecting the expression level of a disintegrin metallopeptidase 8 containing a platelet-derived tuberculin type 1 motif in plasma.

[0011] Furthermore, the diagnostic product is a kit, a reagent or a chip.

[0012] Furthermore, the reagent is an ELISA detection reagent.

[0013] The present invention discloses the following technical effects:

[0014] The present invention screens serum markers for early diagnosis of patients with aortic dissection, and has great clinical value and practical significance for the early diagnosis of acute aortic dissection, long-term prognosis and improvement of quality of life. The present invention screens serum markers for early diagnosis of patients with aortic dissection, and has great clinical value and practical significance for the early diagnosis of acute aortic dissection, long-term prognosis and improvement of quality of life. The present invention obtained the differentially expressed gene ADAMTS8 of aortic dissection through spatial transcriptomics screening, and used it as a molecular marker with greater sensitivity and specificity, and was verified by ELISA, proving that ADAMTS8 may be involved in the pathogenesis of acute aortic dissection, and can be used as an early diagnostic biomarker and a new therapeutic target for acute aortic dissection, which provides a new direction for the clinical early diagnosis and long-term prognosis evaluation of aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Expression of specific genes in the ascending aorta of aortic dissection with different severities; A: Stage 1-mild aortic dissection-specific genes and Spot distribution map, the closer the color is to red, the higher the expression level; B: Stage 1-specific gene functional annotation, different colors represent different pathways; C: Stage 2-moderate aortic dissection-specific genes and Spot distribution map, the closer the color is to red, the higher the expression level; D: Stage 2-specific gene functional annotation, different colors represent different pathways; E: Stage 3-severe aortic dissection-specific genes and Spot distribution map, the closer the color is to red, the higher the expression level; F: Stage 3-specific gene functional annotation, different colors represent different pathways;

[0017] Figure 2 The expression level and concentration of ADAMTS8 as a biomarker in the normal control group and the aortic dissection group (* indicates P < 0.05);

[0018] Figure 3 This is the receiver operating characteristic curve for comparative analysis of ADAMTS8 as a biomarker in the normal control group and the aortic dissection group. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is specifically disclosed. Any intermediate value within a stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Terminology Notes:

[0025] CXCL1: chemokine (CXC motif) ligand 1;

[0026] PLS3: actin 3;

[0027] PTMA: prothymosin α;

[0028] SPARC: secreted protein acidic and rich in cysteine / human osteonectin;

[0029] ADAMTS8: A disintegrin metallopeptidase 8 containing platelet-type 1 tuberculin motif;

[0030] CD36: scavenger receptor B2 / human platelet membrane glycoprotein IV.

[0031] Example 1 Spatial transcriptomics analysis of differentially expressed genes in patients with aortic dissection

[0032] A total of 15 patients with aortic dissection who underwent surgical vascular replacement were collected. After morphological identification of tissue samples by HE staining and RIN testing for tissue RNA quality control, 8 samples were finally included. According to the results of aortic computed tomography imaging, aortic dissection cases were divided into 3 groups of different severity according to the widest diameter of the ascending aorta: 35-40mm, 2 cases; 41-45mm, 4 cases; 46-50mm, 2 cases. A total of 19 slices of samples from all different parts (ascending aorta, brachiocephalic artery, left subclavian artery, left common carotid artery) were sliced. Spatial transcriptomics technology was then used to analyze differentially expressed genes in patients with aortic dissection. All subjects signed written informed consent, and this study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. The specific methods of spatial transcriptomics technology and analysis are as follows:

[0033] 1. Human aorta tissue collection, quick freezing and embedding

[0034] Within 30 minutes of revascularization, lesion tissue collected should be quickly placed in a frozen tissue vial. After removing excess adventitial tissue and blood clots, the tissue should be trimmed to a cross-sectional area of ​​approximately 6 × 6 mm. Using forceps, place the frozen tissue into an appropriately sized embedding cassette. Rapidly embed the tissue sample in OCT embedding gel and place on dry ice until the OCT solidifies and turns white. Transfer the OCT-embedded tissue to a cryostat and equilibrate in a cryostat for at least 20 minutes. Adjust the slice thickness, cut 20 slices, and stack the slices in a labeled, pre-chilled 1.5 mL centrifuge tube.

[0035] 2. RNA extraction, RIN value assessment and patching

[0036] RNA was extracted using the Trizol method and the Qiagen RNeasy Mini kit (Qiagen, Germany). Purified RNA was stored at -80°C for long-term storage or immediately analyzed for RNA number identification (RIN) using an Agilent RNA 6000Nano or Pico kit (Agilent, USA). For specific procedures, refer to the Agilent RNA 6000Nano kit instructions, and analysis was performed according to the Agilent 2100 Bioanalyzer system operating instructions. Samples with an RIN > 7 were considered acceptable. Qualified samples were mounted on Visium spatial gene slides pre-equilibrated in a cryostat.

[0037] 3. Preparation of Spatial Organization Optimization Slides and Spatial Gene Expression Slides

[0038] The Visium spatial gene expression solution applies spatial transcriptomics technology to standard methods of tissue sectioning and staining to study the expression of mRNA in the entire transcriptome at spatial resolution, while obtaining histologically related information in the same tissue section. It is a method for spatial resolution visualization and quantitative analysis of the transcriptome in a single tissue sample. There are four capture areas (6.5×6.5mm) on each slide used for library construction. Each capture area contains 5,000 barcoded spots (Barcoded Spots) with a diameter of 55μm, and each spot has a unique barcode sequence. When RNA is released from the cells in the tissue section, the RNA that migrates to each spot is labeled with the corresponding barcode sequence, and then the library is constructed and sequenced. Afterwards, the data is assigned according to the barcode information of the data to determine the location of the data source, ultimately achieving visualization of spatial gene expression.

[0039] The Spatial Gene Expression Solution Kit includes two slides: the Visium Spatial Gene Tissue Optimization Slide and the Visium Spatial Gene Expression Slide.

[0040] (1) Visium Spatial Gene Tissue Optimization Slide: Used to determine the optimal permeabilization time for specific tissue types. It includes 8 capture zones, each covered with oligonucleotides for mRNA capture. Each capture zone is surrounded by an 8×8mm fiducial frame. The readable label (serial number and QR code) is the active surface of the slide.

[0041] (2) Visium spatial gene expression slides: Generate a Visium spatial gene expression library, which includes 4 capture zones, each 6.5×6.5 mm, with approximately 5000 unique gene expression barcoded spots surrounded by a reference frame with a total area of ​​8×8 mm.

[0042] The primer sequences for the Visium Spatial Gene Expression Slide Active Surface are:

[0043] 5'-CTACACGACGCTCTTCCGATCT-NNNNNNNNNNNNNNNN-NNNNNNNNNNNN-TTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO. 1).

[0044] 4. Organizational optimization

[0045] Before library construction, Visium Spatial Tissue Optimization can optimize the permeabilization conditions of the tissue of interest. Tissue sections are stained with hematoxylin and eosin (HE) and photographed in brightfield using a Leica SCN 400 slide scanner. Fixed and stained tissue sections are then treated with permeabilization enzyme for varying durations (3, 6, 12, 18, 24, and 30 minutes). Tissue patches are then mounted on the capture zone of a Visium Spatial Tissue Optimization slide. The sections, fixed and stained, are then permeabilized for varying durations. During the permeabilization process, mRNA released from the tissue is captured by the capture zone of the slide. A mixture containing reverse transcription reagents and fluorescently labeled nucleotides is added to the surface of the tissue patch to generate fluorescently labeled cDNA. The tissue is then removed with a tissue removal enzyme, leaving the fluorescent cDNA covalently linked to the oligonucleotide on the tissue-optimized slide. The fluorescent cDNA is visualized using a Leica DMi8 fluorescence microscope. Comparison of tissue morphology between brightfield and fluorescent images of HE staining indicates that the optimal permeabilization time yields the maximum fluorescent signal.

[0046] 5. Visium spatial gene expression

[0047] The Visium spatial gene expression solution measures the total mRNA in intact tissue sections and maps the locations of actively expressed genes. Each spatial gene expression slide contains a capture area with gene expression points, which includes primers required to capture mRNA and substrates for polyadenylated mRNA. Tissue sections placed in these capture areas are fixed and stained. After permeabilization with permeabilization enzymes, cellular mRNA is captured by primers on gene expression points. All cDNAs generated by mRNA captured by primers at specific sites have a common spatial barcode. A single-cell 3' gene expression library is generated from cDNA and sequenced. The spatial barcode is used to associate the reads with the tissue section image to perform spatial gene expression mapping. The steps at this stage include the following parts:

[0048] (1) Tissue sectioning, fixation, and staining;

[0049] (2) Tissue permeabilization, reverse transcription, and obtaining cDNA using the optimal conditions screened by "tissue optimization";

[0050] (3) Second-strand synthesis and denaturation: Second-strand synthesis primer: 5'-AAGCAGTGGTATCAACGCAGAG-3' (SEQ ID NO. 3);

[0051] (4) cDNA amplification and quality control:

[0052] The cDNA primers included a forward primer: 5'-CTACACGACGCTCTTCCGATCT-3' (SEQ ID NO. 2) and a reverse primer: 5'-AAGCAGTGGTATCAACGCAGAG-3' (SEQ ID NO. 3).

[0053] The quality of cDNA was checked using an Agilent 2100 Bioanalyzer.

[0054] 6. Construction of Visium spatial gene expression library

[0055] The cDNA obtained in the previous step was used to construct the Visium spatial gene expression library. The steps include the following: (1) Using a PCR instrument to start the fragmentation program to fragment the sample; (2) After fragmentation, end repair, A tail addition, and SPRI double-end screening of the sample using the SPRI screening kit (Beckman Coulter); (3) Adapter ligation; (4) After adapter ligation, purification and SPRI screening; (5) Sample addition of double-end index and PCR; (6) After the sample addition of double-end index, PCR double-end SPRI screening; (7) Quality control after library construction.

[0056] 7. Sequencing

[0057] Visium spatial gene expression libraries are standard Illumina paired sequences starting with P5 and ending with P7. Sequencing these libraries generates a standard Illumina BCL output data file.

[0058] (1) Sequencing depth estimation: Calculated based on the area of ​​the capture region covered by tissue HE staining bright field imaging, total sequencing depth = [capture area (%) × total number of spots in the capture region (5000)] × 50,000 readpairs / spot

[0059] (2) The library was sequenced by Shanghai Jingneng Biotechnology Co., Ltd. using the IluminaNova 6000 sequencing platform.

[0060] 8. Data bioinformatics analysis

[0061] Raw data were processed using the official 10×Genomics analysis pipeline and software. Quality control methods and a data analysis pipeline (SCTransform normalization, PCA dim30, t-SNE, and UMAP) were used to analyze the raw data obtained after sequencing. A detailed analysis of gene expression and cell types was performed across different aortic sites and ascending aorta severity levels in patients with aortic dissection. A gene expression and cell atlas for aortic dissection was established.

[0062] Analysis of differentially expressed genes in different severity levels of ascending aorta: A sample of different severity levels of ascending aorta was randomly selected for analysis. Seurat was used to perform differentially expressed gene analysis, including shared differentially expressed genes and differentially expressed genes specific to each sample with different severity levels. Metascape software was then used for functional annotation, and several genes related to spatial location were selected to display their distribution.

[0063] 9. Gene expression patterns in ascending aorta with different severity levels in aortic dissection

[0064] Results: Analysis of gene expression in the ascending aorta at different stages of aortic dissection showed that there were 103 genes specific to stage 1-mild aortic dissection, 72 genes specific to stage 2-severe aortic dissection, and 268 genes specific to stage 3-severe aortic dissection. There were 181 genes commonly expressed in the three stages.

[0065] Among them, the specific gene expression patterns of different severity levels of ascending aorta in aortic dissection are as follows: Stage 1 - mild aortic dissection. Two specific genes were screened based on gene expression location, including CXCL1 and PLS3, which are mainly distributed in the ascending aorta intima, and the number of spots was visualized (see Figure 1 A), functional annotations were performed on these two genes, showing the top 20 pathways involved. They were mainly involved in interleukin-mediated signaling pathways and inflammatory factor signaling pathways, and were related to inflammatory responses (see Figure 1 Middle B); In stage 2—moderate aortic dissection, two unique genes, including PTMA and SPARC, were screened based on gene expression location. They were mainly distributed in the tear of the ascending aorta media, and the number of spots was visualized (see Figure 1 C), functional annotation of these two genes was performed, showing the top 20 pathways involved, which are mainly involved in the maintenance of extracellular matrix structure and organization, bone development, and vascular development pathways, which are related to vascular remodeling (see Figure 1 Middle D); Similarly, in stage 3-severe aortic dissection, two unique genes, ADAMTS8 and CD36, were screened based on gene expression location. They were mainly distributed in the adventitia of the ascending aorta, and the number of spots was visualized (see Figure 1 Middle E), functional annotation of these two genes was performed, showing the top 20 pathways involved, which mainly participated in cell adhesion regulation and hypoxia signaling pathways, which are related to cell activity and oxygen homeostasis (see Figure 1 Middle F).

[0066] Example 2 Validation of expanded sample of serum markers in patients with aortic dissection

[0067] 1. Materials and Methods

[0068] Based on inclusion and exclusion criteria, 131 patients diagnosed with aortic dissection by aortic CT angiography and 137 healthy controls were enrolled. Peripheral blood plasma was collected from these patients for ELISA testing to verify the ability of ADAMTS8 to identify patients with aortic dissection. The ADAMTS8 ELISA kit (JL14480, Shanghai Jianglai Co., Ltd., China) was used. Patients with aortic dissection were included if they had a confirmed diagnosis of acute aortic dissection by aortic CT angiography and were ≥18 years of age and had signed informed consent. Exclusion criteria included patients with a history of rheumatic disease, autoimmune disease, aortitis, pregnancy, or hereditary syndromes (such as Marfan syndrome). Healthy controls were included if they were ≤65 years of age undergoing a physical examination, had no hypertension or medication-controlled hypertension, no history of cardiovascular disease, and had coronary artery disease excluded by coronary angiography. Exclusion criteria included patients with comorbid cardiovascular disease or systemic disease.

[0069] The experimental principle is to use a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled detection antibodies, and HRP enzyme conjugates are added sequentially to microwells pre-coated with the corresponding index capture antibody. After incubation and washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. The depth of the color is positively correlated with the concentration of the corresponding protein in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using a microplate reader to calculate the sample concentration. For detailed experimental steps, please refer to the kit instructions.

[0070] Things to note are as follows:

[0071] (1) Sample processing: Plasma is used for testing. Specimens are collected using EDTA or heparin as an anticoagulant. The specimens are centrifuged at 1000×g for 15 minutes at 2°C within 30 minutes after collection. The supernatant can be taken for testing, or the supernatant can be stored at -20°C (or -80°C). However, repeated freezing and thawing should be avoided.

[0072] (2) Preparation of standard gradient working solution: Prepare the standard gradient working solution in EP tubes using the serial dilution method. Please refer to the instructions of each kit for details.

[0073] (3) Prepare biotinylated antibody working solution, enzyme conjugate working solution and 1× washing working solution in advance.

[0074] (4) Result evaluation: ① Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. Plot the standard curve of the four-parameter logistic function on double-logarithmic coordinate paper with concentration as the horizontal axis and OD value as the vertical axis (remove the blank group value when plotting). ② If the sample OD value is higher than the upper limit of the standard curve, it should be diluted appropriately and re-measured and the corresponding dilution factor should be multiplied when calculating the sample concentration.

[0075] The aortic dissection group and the normal healthy group were analyzed and detected using the above-mentioned ELISA method using ADAMTS8 as a biomarker. The obtained data were analyzed using SPSS 26.0 statistical software, and the receiver operating characteristic (ROC) curve was drawn to determine whether the new marker could become a new diagnostic marker for aortic dissection.

[0076] 2. Results

[0077] like Figure 2 As shown in Table 1, the figure shows the expression level of ADAMTS8 as a biomarker in the normal control group and the aortic dissection group. The results show that ADAMTS8 can well distinguish healthy people from aortic dissection patients.

[0078] Table 1 ADAMTS8 plasma ELISA concentration

[0079]

[0080] The receiver operating characteristic curve of ADAMTS8 as a biomarker in distinguishing the normal control group from the aortic dissection group, as shown in Figure 3 and as shown in Table 2. The results showed that the area under the receiver operating characteristic curve was greater than 0.7, which had a good diagnostic value for aortic dissection.

[0081] Table 2 ROC analysis of blood ADAMTS8 for screening aortic dissection

[0082]

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of a disintegrin metallopeptidase 8 containing a platelet tuberculin type 1 motif in plasma in the preparation of a diagnostic product for acute aortic dissection.

2. The use according to claim 1, characterized in that The diagnostic product is a kit, a reagent or a chip.

3. The use according to claim 1, characterized in that The reagent is an ELISA detection reagent.

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