Vascular plaque diagnostic kit based on NT-IGFBP-4 and GDF-15 detection

Abstract

The invention discloses an application of NT-IGFBP-4 and GDF-15 as biomarkers in preparation of a diagnostic reagent for clinical events related to vascular plaques. Or the NT-IGFBP-4 and / or GDF-15 are / is used as a drug target to develop a drug for treating vascular plaque. When the NT-IGFBP-4 level in a biological sample is higher than the average level of healthy individuals by 13.8% or above and the NT-IGFBP-4 level in serum reaches 119.2 ng / mL or above; or when the GDF-15 level is higher than the average level of healthy individuals by 45.2% or above and the serum GDF-15 level reaches 1221 pg / mL or above, the blood vessel plaque related clinical event is basically judged; the substance for inhibiting the expression of NT-IGFBP-4 and / or GDF-15 can be used as a candidate drug for treating vascular plaque.

Description

Technical Field

[0001] This invention belongs to the field of biological detection and relates to a method for detecting and diagnosing vascular plaque-related clinical events based on NT-IGFBP-4 and GDF-15 levels, either alone or in combination. Background Technology

[0002] Atherosclerotic plaques are a common chronic disease that seriously threatens human health worldwide. The formation of plaques and the resulting vascular stenosis are important indicators for clinically assessing the severity of vascular disease. Most patients eventually develop plaque-related clinical events such as stable angina, unstable angina, and acute myocardial infarction, based on the underlying coronary atherosclerotic plaque stenosis. However, the early progression of plaque events is mainly characterized by functional cellular cascade inflammatory responses, with little impact on hemodynamics, and the symptoms are often subtle and difficult to detect.

[0003] The diagnosis of atherosclerotic plaques is currently primarily achieved through imaging techniques such as ultrasound, CT, and MRI. While these methods provide direct information about the plaques, they suffer from limitations including a single diagnostic approach, complex procedures, and high costs, hindering broad-based screening. Furthermore, the process is susceptible to the subjective biases and experience of individual physicians, making it difficult to sensitively detect early-stage or occult plaques. In addition, current technologies lack efficient and sensitive biomarkers for early detection and accurate diagnosis. Existing hematological indicators, such as C-reactive protein, blood lipids, or troponin, primarily reflect systemic inflammation or myocardial damage, offering limited effectiveness in identifying plaque ischemia and failing to meet the needs of plaque event detection.

[0004] The patent document with publication number CN120126753A reports some compounds that may be used as biomarkers for the diagnosis of carotid artery plaques. These compounds include lipoxygenin A4, ethyl 3-indole, N-(3-indoleacetyl)-L-alanine, phosphatidylserine PS (19:0-18:2), phosphatidic acid PA (20:0-21:0), cholesterol ester CE (19:0), lysophosphatidylcholine LPC (O-22:1), and phosphatidylethanolamine PE (P-17:0-18:1). In practice, it is expensive to use liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) to detect the content of all these compounds, and then use complex mathematical models to calculate the risk of carotid artery plaques, which is difficult for ordinary hospitals to afford and implement. The patent document also discloses several proteins that can be used as biomarkers, including thrombomodulin, intercellular adhesion molecule ICAM-3, P-selectin, growth differentiation factor-15GDF-15, adiponectin, monocyte chemotactic protein-1MCP-1, interleukin-10IL-10, placental growth factor 1GF, epidermal growth factor-like tyrosine kinase receptor 2Tie-2, vascular endothelial growth factor D VEGF-D, and vascular cell adhesion molecule 1VCAM-1. In practice, expensive detection devices are needed to detect the expression levels of all these proteins, and then complex mathematical models are used to calculate and determine the risk of carotid artery plaques. Due to the high cost of detection, this approach is not practical at all.

[0005] Therefore, identifying and establishing diagnostic biomarkers for plaque events is of great clinical significance, and can help provide effective targets and new ideas for the early clinical diagnosis and treatment of atherosclerotic plaques and improve prognosis. Summary of the Invention

[0006] To develop a practical, simple, and efficient method for detecting vascular plaque-related clinical events that can be readily implemented in general hospitals and even general departments, we explored the use of one or two biomarkers for the early identification of vascular plaque-related clinical events (including but not limited to stable angina, unstable angina, and acute myocardial infarction) in our research on the pathogenesis of vascular plaques, especially arterial plaques. Through a systematic investigation of genes, RNA, and proteins with abnormal expression (significantly increased or decreased expression) in patients, we identified N-terminal insulin-like growth factor binding protein-4 (NT-IGFBP-4) and growth differentiation factor-15 (GDF-15) as single-marker and combined diagnostic methods. This method has advantages such as low invasiveness, repeatability, and ease of screening, significantly improving diagnostic efficacy and sensitivity, thereby achieving accurate screening and early intervention for vascular plaque-related clinical events. Based on these findings, this invention includes the following technical solutions:

[0007] This invention primarily provides the application of N-terminal insulin-like growth factor binding protein-4 (NT-IGFBP-4) and / or growth differentiation factor-15 (GDF-15) as biomarkers in the preparation of diagnostic reagents for vascular plaque-related clinical events; or the use of NT-IGFBP-4 and / or GDF-15 as drug targets in the development of drugs for the treatment of vascular plaques.

[0008] In one embodiment, the aforementioned vascular plaque refers to arterial plaque, particularly coronary artery plaque.

[0009] The aforementioned vascular plaque-related clinical events refer to clinically significant endpoint events triggered or associated with atherosclerotic plaques, primarily including, but not limited to, stable angina, unstable angina, and acute myocardial infarction (including ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction). These events are direct clinical manifestations of decreased plaque stability and rupture.

[0010] The NT-IGFBP-4 level in the above biological samples refers to the concentration / content of NT-IGFBP-4 protein, or the gene / mRNA expression level of NT-IGFBP-4; the GDF-15 level in the biological samples refers to the concentration / content of GDF-15 protein, or the gene / mRNA expression level of GDF-15.

[0011] The biological samples mentioned above can be selected from the following group: whole blood, plasma, serum, lung tissue samples, tissue fluid, saliva, oral mucosa, nasopharyngeal secretions, body fluids, and urine, with serum being the preferred biological sample.

[0012] In one biomarker application approach, when the level / concentration of NT-IGFBP-4 in a biological sample is more than 13.8% higher than the average level of healthy individuals, and the serum NT-IGFBP-4 level reaches more than 119.2 ng / mL; or / and the level / concentration of GDF-15 is more than 45% higher than the average level of healthy individuals, and the serum GDF-15 level reaches more than 1221 pg / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event; substances that inhibit the expression of NT-IGFBP-4 and / or GDF-15 can serve as candidates for vascular plaque treatment, especially arterial plaque treatment drugs.

[0013] Preferably, the reagents are kits, specifically NT-IGFBP-4 and / or GDF-15 detection kits for diagnosing vascular plaque-related clinical events, especially arterial plaque endpoint events.

[0014] When the above kit is used to detect the gene / mRNA expression levels of NT-IGFBP-4 and / or GDF-15 in biological samples, the kit detects the gene levels of NT-IGFBP-4 and / or GDF-15 in biological samples via qRT-PCR, including: commonly used biochemical reagents for qRT-PCR reactions to detect the mRNA content / level of the NT-IGFBP-4 gene in biological samples; and commonly used biochemical reagents for qRT-PCR reactions to detect the mRNA content / level of the GDF-15 gene in biological samples.

[0015] Furthermore, the kit also includes: an RNA extraction and separation system for extracting and isolating total RNA from biological samples; a reverse transcription system for reverse transcribing total RNA into cDNA; and primers for detecting the NT-IGFBP-4 and / or GDF-15 genes and internal reference genes.

[0016] In another embodiment, the above kit is used to detect the concentration / content of NT-IGFBP-4 and / or GDF-15 proteins in biological samples, and is a latex immunoturbidimetric assay kit, a magnetic microparticle chemiluminescence assay kit, a radioimmunoassay kit, or an immunochromatographic test strip.

[0017] For example, the above kit is a double-antibody sandwich kit, which can be selected from the following groups of methods:

[0018] A. Enzyme-linked immunosorbent assay kit;

[0019] B. Magnetic microparticle chemiluminescence assay kit;

[0020] C. Immunochromatographic test strips.

[0021] Preferably, any of the above-mentioned reagent kits also includes an instruction manual.

[0022] The instructions can be written on bottles, test tubes and similar objects, plates, or on a separate piece of paper, or on the outside or inside of a container, such as a paper document with an operation demonstration video app download window or a QR code. The instructions can also be in multimedia form, such as a CD, USB flash drive, or cloud storage.

[0023] Furthermore, the above-mentioned kit also includes a carrier containing mathematical models for diagnosing clinical events related to vascular plaques, said mathematical models being divided into three categories:

[0024] First, the single biomarker NT-IGFBP-4 or GDF-15 is used for judgment. When the NT-IGFBP-4 level is more than 13.8% higher than the average level of healthy individuals and the serum NT-IGFBP-4 level reaches more than 119.2 ng / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event. Alternatively, when the GDF-15 level / concentration is more than 45% higher than the average level of healthy individuals and the serum GDF-15 level reaches more than 1221 pg / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event.

[0025] Second, the binary logistic regression model for joint / combined judgment of NT-IGFBP-4 and GDF-15 dual biomarkers is shown in the following formula:

[0026] Joint inspection parameters = -15.982 + 5.694 × [NT-IGFBP-4] + 1.891 × [GDF-15]

[0027] In the formula, [NT-IGFBP-4] is the serum detection concentration of NT-IGFBP-4, [GDF-15] is the serum detection concentration of GDF-15, and the cut-off value is 0.7204. The [NT-IGFBP-4] and [GDF-15] values ​​obtained from the detection are entered into the formula for calculation. When the calculated value is above 0.7204, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event.

[0028] Thirdly, the decision tree model for the combined / integrated judgment of dual biomarkers NT-IGFBP-4 and GDF-15 has a serum detection concentration threshold (Cut-off) of firstly GDF-15 not less than 897.00 pg / mL and then NT-IGFBP-4 not less than 90.4 ng / mL. When the detected values ​​of [NT-IGFBP-4] and [GDF-15] are greater than this threshold, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event.

[0029] In one embodiment of drug target application, the vascular plaque treatment drug is an NT-IGFBP-4 and / or GDF-15 gene expression inhibitor / antagonist.

[0030] The aforementioned drugs for treating vascular plaques can be chemical drugs such as small molecule compounds, or biological drugs such as protein drugs or nucleic acid drugs. For example, RNAi molecules are siRNA molecules or shRNA molecules that target the NT-IGFBP-4 and / or GDF-15 genes.

[0031] This invention novelly discovers that NT-IGFBP-4 and GDF-15 can be used alone as biomarkers for the auxiliary diagnosis and early screening of vascular plaque-related clinical events. By detecting the concentration levels of NT-IGFBP-4 and GDF-15 in the blood of subjects, either alone or in combination, and combining this with reference thresholds or combined diagnostic models, the diagnostic sensitivity and positive predictive value can be improved. This facilitates the early identification, risk stratification, prognostic assessment, and clinical triage of plaque-related ischemic events, and provides a reference for disease monitoring and efficacy evaluation. This method is simple, rapid, and highly practical, and has significant clinical significance and widespread application value for the diagnosis and treatment of vascular plaques, especially arterial plaques. Attached Figure Description

[0032] Figure 1 The concentration distribution of NT-IGFBP-4 and GDF-15 in healthy individuals and individuals with vascular plaque-related clinical events is shown.

[0033] Figure 2 The ROC curves of NT-IGFBP-4 alone for the diagnosis of vascular plaque-related clinical events are shown.

[0034] Figure 3 The ROC curve of GDF-15 alone for the diagnosis of vascular plaque-related clinical events is shown.

[0035] Figure 4 The ROC curves of NT-IGFBP-4 and GDF-15 in combination for the combined diagnosis of vascular plaque-related clinical events are shown.

[0036] Figure 5 A decision tree model for joint diagnosis using NT-IGFBP-4 and GDF-15 is shown. Detailed Implementation

[0037] Currently, the diagnosis of clinical events related to vascular plaques mainly relies on imaging examinations, which have limitations such as complex procedures, high costs, and difficulty in achieving early screening. Existing blood biomarkers, such as C-reactive protein and troponin, have limited ability to identify plaque ischemia. In our study on the pathogenesis of arterial plaques, we screened out two biomarkers, NT-IGFBP-4 and GDF-15. NT-IGFBP-4 is the N-terminal fragment of IGFBP-4 mediated by pregnancy-associated plasma protein-A and is closely related to plaque activity, myocardial remodeling, and adverse cardiovascular events. GDF-15 belongs to the TGF-β superfamily and is highly expressed in myocardial and vascular endothelial cells during stress, inflammation, or injury. Since both are involved in the pathological processes of atherosclerosis and ischemia, and their concentrations are significantly elevated in plaque event populations, NT-IGFBP-4 and / or GDF-15 can be used alone or in combination. By leveraging their biological correlation and complementarity, a more sensitive and specific diagnostic method can be constructed, making up for the shortcomings of existing technologies and achieving non-invasive, early, and reliable risk assessment and auxiliary diagnosis of plaque endpoint events.

[0038] To achieve simplicity and rapid practicality, the detection of NT-IGFBP-4 and / or GDF-15 can be performed using a kit. Those skilled in the art will readily understand that, in addition to commonly used biological and chemical reagents for detecting NT-IGFBP-4 and / or GDF-15, the kit of this invention may also include at least one of the following: a carrying tool, the space of which is divided into defined spaces capable of accommodating one or more containers, 96-well plates, or strips, such as kits, vials, test tubes, and the like, each containing a single component for the method of this invention; and an instruction manual, which may be written on the vials, test tubes, and the like, or on a separate sheet of paper, or on the outside or inside of the container, for example, a paper document with an operation demonstration video / APP download window such as a QR code. The instruction manual may also be in a tangible or intangible multimedia form, such as a USB flash drive or cloud storage.

[0039] In the description of the technical solution of the present invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone).

[0040] When the kit is used to detect whether a subject has experienced a plaque-related clinical event (including stable angina, unstable angina and acute myocardial infarction), the detection process includes, for example, the following steps: Step 1, collect a subject sample; Step 2, detect the serum concentration levels of NT-IGFBP-4 and / or GDF-15 proteins; Step 3, select one of the following methods to output the diagnostic results: (1) Single marker diagnosis: compare the individual detection concentration of NT-IGFBP-4 or GDF-15 with the reference value, i.e., the cutoff standard (threshold); (2) Combined diagnosis: input the NT-IGFBP-4 and GDF-15 concentration data into the combined diagnostic model for judgment. The combined diagnostic model may use binary logistic regression, decision tree or other suitable algorithms to improve the required corresponding diagnostic performance.

[0041] Step 2 may include: Step 2-1, detecting the expression level of NT-IGFBP-4 using chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), or other specific immunoassay methods; Step 2-2, detecting the expression level of GDF-15 using chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), or other specific immunoassay methods.

[0042] Step 3 may include: Step 3-1, in the case of single-marker diagnosis, comparing the detection concentration level of NT-IGFBP-4 with a reference value, i.e., a cutoff value (threshold). If the detection concentration level of NT-IGFBP-4 is higher than the reference value, it indicates that the subject is diagnosed with a positive vascular plaque-related clinical event; if the detection concentration level of NT-IGFBP-4 is lower than the reference value, it indicates that the subject is diagnosed with a negative vascular plaque-related clinical event. Step 3-2, in the case of single-marker diagnosis, comparing the detection concentration level of GDF-15 with a reference value, i.e., a cutoff value (threshold). If the detection concentration level of GDF-15 is higher than the reference value, it indicates that the subject is diagnosed with a positive vascular plaque-related clinical event; if the detection concentration level of GDF-15 is lower than the reference value, it indicates that the subject is diagnosed with a negative vascular plaque-related clinical event. Step 3-3: In the case of joint diagnosis, output the detection concentration levels of NT-IGFBP-4 and GDF-15 into the joint diagnostic model (binary logistic regression, decision tree or other suitable algorithm model), and obtain the final diagnostic result based on the model reference value, i.e. the boundary standard (threshold).

[0043] On the other hand, given the positive correlation between the expression levels of the biomarkers NT-IGFBP-4 and GDF-15 and vascular plaque-related clinical events, NT-IGFBP-4 and GDF-15 may serve as screening targets for vascular plaque treatment drugs. That is, gene inhibitors / antagonists that reduce the expression levels of NT-IGFBP-4 and GDF-15 could be candidates for vascular plaque treatment drugs.

[0044] As used in this article, “(NT-IGFBP-4 and / or GDF-15 expression levels) decreased,” “decreased,” or “downregulated” can mean a decrease of at least 10% compared to a reference level (e.g., the original level), such as a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50% compared to a reference level, until it is reduced to the average level of healthy individuals or the vascular plaque disappears (coronary artery stenosis <30%).

[0045] As for drugs for treating vascular plaques, they can be chemical drugs or biological products, including but not limited to small molecule compounds, protein drugs or nucleic acid drugs such as RNAi molecules that cause silencing of the NT-IGFBP-4 and / or GDF-15 genes.

[0046] In one embodiment, the drug may be a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the therapeutic drug as described above, also contains one or more other pharmaceutical ingredients that inhibit the expression of NT-IGFBP-4 and / or GDF-15.

[0047] In another embodiment, the above-mentioned drug is a pharmaceutical composition that, in addition to containing a therapeutically effective amount of the therapeutic drug as described above, also contains one or more other pharmaceutical ingredients for treating vascular plaques.

[0048] Compared with existing vascular plaque diagnostic technologies, the positive effects of this invention include at least the following aspects:

[0049] 1. Convenient, non-invasive and easy to promote: Based on blood sample detection of up to two components, NT-IGFBP-4 and GDF-15, no complex imaging equipment is required. It is simple to operate, low in cost, and suitable for medical institutions at all levels and large-scale population screening.

[0050] 2. Early identification and high sensitivity: NT-IGFBP-4 and GDF-15 are significantly elevated in the early stage of plaque events. Combined detection can significantly improve diagnostic sensitivity (up to 93% in Example 4), which helps to achieve early detection of occult lesions.

[0051] 3. It has good diagnostic efficacy and reliability: the AUC of the combined diagnostic model can reach 0.716, which is better than the detection of individual biomarkers, and the positive predictive value is high (such as 91% in the decision tree model), making the results more clinically valuable.

[0052] 4. Flexible and adaptable model: Supports multiple diagnostic strategies such as single marker determination, binary logistic regression, and decision tree. The model can be selected or adjusted according to actual needs to enhance the applicability and scalability of the method.

[0053] 5. Strong complementarity of biomarkers: NT-IGFBP-4 and GDF-15 reflect plaque activity and systemic / vascular stress status, respectively. Their combined use can improve the comprehensive judgment of plaque ischemic events from multiple perspectives.

[0054] 6. Facilitates integration with existing clinical pathways: It can be used in conjunction with symptoms, imaging and other laboratory indicators to assist in clinical stratification, efficacy monitoring and prognostic assessment, and promote the implementation of precision medicine.

[0055] In summary, this invention significantly improves the identification and diagnostic accuracy of vascular plaque-related clinical events while maintaining convenient detection, and has high potential for clinical translation and application.

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0057] Example

[0058] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0059] The commercial reagent kits used in the examples were operated according to their instructions.

[0060] All blood samples from patients with vascular plaques and from healthy individuals were obtained from the Department of Pathology and Department of Cardiology at Zhongshan Hospital affiliated with Fudan University, as well as from healthy volunteers. Among them, 254 cases involved patients with vascular plaque-related clinical events, and 51 cases involved healthy individuals.

[0061] Example 1

[0062] This embodiment analyzes the differences in concentration levels of NT-IGFBP-4 and GDF-15 between healthy individuals and individuals with vascular plaque-related clinical events.

[0063] To evaluate the application of NT-IGFBP-4 and GDF-15 in detecting vascular plaque-related clinical events, 51 healthy individuals (plaque stenosis <30%) and 254 individuals experiencing vascular plaque-related clinical events (including unstable angina, stable angina, and acute myocardial infarction) were included. All subjects met the following common criteria: age >18 years, underwent coronary angiography, and signed informed consent; those with active autoimmune diseases, active malignancies, severe hepatic or renal insufficiency, or recent major trauma / surgery were excluded. In healthy individuals, angiography showed all major coronary artery stenosis <30% (including completely normal arteries). Based on coronary angiography and clinical diagnosis, the population with vascular plaque-related clinical events was divided into the following three groups: (1) Stable angina group: at least one major coronary artery with ≥30% stenosis, typical stable angina symptoms (CCS grade I-III), and troponin (cTnI / T) level not exceeding the upper limit of normal; no history of stent implantation; (2) Unstable angina group: at least one major coronary artery with ≥30% culprit lesion, clinical manifestations conforming to the definition of unstable angina (such as first-onset, worsening, or resting angina), and troponin not exceeding the upper limit of normal; no history of stent implantation; (3) Acute myocardial infarction group (including STEMI and NSTEMI): culprit lesion confirmed by coronary angiography, and troponin level exceeding the upper limit of normal. All diagnoses were in accordance with current international guidelines. Blood was collected from both groups upon admission using vacuum blood collection tubes, and serum was obtained by centrifugation after standing at room temperature. The samples were processed and stored at -80℃ until testing. The biomarkers were detected using a fully automated chemiluminescence immunoassay analyzer CL-8000i (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), and the concentration levels of biomarkers NT-IGFBP-4 and GDF-15 were measured, respectively. Statistical analysis was performed using Graph Pad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and IBM SPSS Statistics 27.0 (SPSS Inc., Chicago, IL, USA) software. The Mann-Whiney test (for non-normal distribution) was used to compare continuous data between groups. A p-value < 0.05 was considered statistically significant.

[0064] The results are as follows Figure 1As shown, the concentration levels of NT-IGFBP-4 and GDF-15 differed significantly between healthy individuals and those experiencing vascular plaque-related clinical events (p < 0.0001). Specifically, the median and quartiles of NT-IGFBP-4 in healthy individuals were 110.9 (89.0–127.6) ng / mL, while these significantly increased to 126.2 (107.8–146.0) ng / mL in the vascular plaque-related clinical event population. Similarly, the median and quartiles of GDF-15 in healthy individuals were 990.13 (768.92–1507.86) pg / mL, while these significantly increased to 1437.63 (1043.73–2139.57) pg / mL in the vascular plaque-related clinical event population. Both NT-IGFBP-4 and GDF-15 are significantly associated with the occurrence of plaque events, suggesting their important application value in the diagnosis of vascular plaque-related clinical events.

[0065] Example 2

[0066] This embodiment is used to evaluate the standalone diagnostic value of NT-IGFBP-4 in vascular plaque-related clinical events.

[0067] This embodiment included 51 healthy individuals (plaque stenosis <30%) and 254 individuals with vascular plaque-related clinical events (including unstable angina, stable angina, and acute myocardial infarction). The specific inclusion and exclusion criteria were consistent with those described in Example 1. Blood samples were collected from both groups upon admission using vacuum blood collection tubes. After standing at room temperature, the blood was centrifuged to obtain serum. Samples were processed and stored at -80°C until testing. The biomarker NT-IGFBP-4 concentration was detected using a fully automated chemiluminescence immunoassay analyzer CL-8000i (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.). Statistical analysis was performed using Graph Pad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and IBM SPSS Statistics 27.0 (SPSS Inc., Chicago, IL, USA) software to calculate the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The Youden index (the sum of sensitivity and specificity minus 1) is used as an indicator to comprehensively evaluate diagnostic efficacy. A higher index indicates stronger diagnostic ability at that threshold; the threshold corresponding to the highest index is considered the optimal diagnostic threshold. A two-sided significance level of p < 0.05 is considered statistically significant.

[0068] Figure 2Table 1 shows the standalone diagnostic performance of NT-IGFBP-4 in vascular plaque-related clinical events. Its area under the ROC curve (AUC) and its 95% confidence interval were 0.680 (0.594-0.765), and its statistical significance was p<0.001. When the Youden index reached its maximum value, the corresponding diagnostic threshold was 119.2 ng / mL, at which point the sensitivity and specificity were 62% and 69%, respectively, and the positive predictive value and negative predictive value were 67% and 65%, respectively.

[0069] Table 1. Diagnostic thresholds and performance of NT-IGFBP-4 for vascular plaque-related clinical events.

[0070]

[0071] Example 3

[0072] This embodiment is used to evaluate the standalone diagnostic value of GDF-15 in vascular plaque-related clinical events.

[0073] This embodiment included 51 healthy individuals (plaque stenosis <30%) and 254 individuals with vascular plaque-related clinical events (including unstable angina, stable angina, and acute myocardial infarction). The specific inclusion and exclusion criteria were consistent with those described in Example 1. Blood samples were collected from both groups upon admission using vacuum blood collection tubes. After standing at room temperature, the blood was centrifuged to obtain serum. Samples were processed and stored at -80°C until testing. The biomarker GDF-15 concentration was detected using a fully automated chemiluminescence immunoassay analyzer CL-8000i (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.). Statistical analysis was performed using Graph Pad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and IBM SPSS Statistics 27.0 (SPSS Inc., Chicago, IL, USA) software to calculate the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The Youden index (the sum of sensitivity and specificity minus 1) is used as an indicator to comprehensively evaluate diagnostic efficacy. A higher index indicates stronger diagnostic ability at that threshold; the threshold corresponding to the highest index is considered the optimal diagnostic threshold. A two-sided significance level of p < 0.05 is considered statistically significant.

[0074] Figure 3Table 2 shows the standalone diagnostic performance of GDF-15 in vascular plaque-related clinical events. Its area under the ROC curve (AUC) and its 95% confidence interval were 0.675 (0.592-0.758), and its statistical significance was p<0.001. When the Youden index reached its maximum value, the corresponding diagnostic threshold was 1221.00 pg / mL, at which point the sensitivity and specificity were 67% and 65%, respectively, and the positive predictive value and negative predictive value were 65% and 66%, respectively.

[0075] Table 2. Diagnostic thresholds and performance of GDF-15 for vascular plaque-related clinical events.

[0076]

[0077] Example 4

[0078] This embodiment is used to evaluate the combined diagnostic value of NT-IGFBP-4 and GDF-15 based on a binary logistic regression model in vascular plaque-related clinical events.

[0079] This embodiment included 51 healthy individuals (plaque stenosis <30%) and 254 individuals with vascular plaque-related clinical events (including unstable angina, stable angina, and acute myocardial infarction). The specific inclusion and exclusion criteria were consistent with those described in Example 1. Blood samples were collected from both groups upon admission using vacuum blood collection tubes. After standing at room temperature, the blood was centrifuged to obtain serum. Samples were processed and stored at -80°C until testing. The biomarker GDF-15 concentration was detected using a fully automated chemiluminescence immunoassay analyzer CL-8000i (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.). Statistical analysis was performed using Graph Pad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and IBM SPSS Statistics 27.0 (SPSS Inc., Chicago, IL, USA) software to calculate the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The Youden index (the sum of sensitivity and specificity minus 1) is used as an indicator to comprehensively evaluate diagnostic efficacy. A higher index indicates stronger diagnostic ability at that threshold; the threshold corresponding to the highest index is considered the optimal diagnostic threshold. A two-sided significance level of p < 0.05 is considered statistically significant.

[0080] In this embodiment, NT-IGFBP-4 and GDF-15 are used as joint diagnostic variables for input, and "whether a vascular plaque-related clinical event occurs" (yes / no) is used as the dependent variable to construct a binary logistic regression model, which outputs the predicted probability of the subject experiencing a vascular plaque-related clinical event. The binary logistic regression model is as follows (the parameters are obtained during the model building phase; this embodiment is only example data):

[0081] Joint inspection parameters = -15.982 + 5.694 × [NT-IGFBP-4] + 1.891 × [GDF-15]

[0082] Wherein, [NT-IGFBP-4] is the detection concentration of NT-IGFBP-4, and [GDF-15] is the detection concentration of GDF-15.

[0083] By inputting the detected biomarker values ​​into the joint detection parameter model for calculation, the diagnostic result can be output based on the threshold.

[0084] Figure 4 Table 3 shows the combined diagnostic performance of NT-IGFBP-4 and GDF-15 in vascular plaque-related clinical events. The area under the ROC curve (AUC) and its 95% confidence interval were 0.716 (range 0.636-0.796), with statistical significance (p < 0.001). When the Youden index reached its maximum value, the diagnostic threshold for the corresponding combined detection model parameters was 0.7204, at which point the sensitivity and specificity were 93% and 65%, respectively, and the positive and negative predictive values ​​were 89% and 55%, respectively. In the combined diagnostic scenario, compared to the individual diagnosis of the biomarkers, the diagnostic sensitivity and positive predictive value were significantly improved. This allows for the timely identification of the vast majority of individuals with actual vascular plaque risk. Furthermore, the probability of 89% of patients diagnosed positive by the combined biomarker diagnosis actually experiencing vascular plaque-related clinical events was high, indicating that the positive results of the biomarkers have high predictive reliability and contribute to early disease identification and rapid triage.

[0085] Table 3. Thresholds and performance of the combined diagnostic model of NT-IGFBP-4 and GDF-15 for vascular plaque-related clinical events (binary logistic regression).

[0086]

[0087] Example 5

[0088] This embodiment is used to evaluate the combined diagnostic value of NT-IGFBP-4 and GDF-15 based on a decision tree model in vascular plaque-related clinical events.

[0089] This embodiment included 51 healthy individuals (plaque stenosis <30%) and 254 individuals with vascular plaque-related clinical events (including unstable angina, stable angina, and acute myocardial infarction), with the specific inclusion and exclusion criteria consistent with those described in Example 1. Blood samples were collected from both groups upon admission using vacuum blood collection tubes. After standing at room temperature, the blood was centrifuged to obtain serum. Samples were processed and stored at -80°C until testing. The biomarker GDF-15 concentration was detected using a fully automated chemiluminescence immunoassay analyzer CL-8000i (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.). IBM SPSS Statistics 27.0 software (SPSS Inc., Chicago, IL, USA) was used to statistically analyze the concentrations of NT-IGFBP-4 and GDF-15 in the subjects through a decision tree analysis module, establishing a decision tree model for the diagnosis of vascular plaque-related clinical events.

[0090] In this embodiment, the concentrations of NT-IGFBP-4 and GDF-15 obtained from the subject samples were input into the SPSS decision tree analysis module, with "whether a vascular plaque-related clinical event occurred" (yes / no) as the dependent variable. A classification decision tree method was used, and the algorithm automatically selected the optimal splitting node for stratification, resulting in the final tree structure. The splitting threshold for each node was calculated using SPSS statistical analysis to ensure maximum information gain for each branch. The resulting decision tree structure is as follows: Figure 5 As shown, the positive and negative results of vascular plaque-related clinical events are determined based on the joint diagnostic decision tree.

[0091] First, enrolled patients were grouped according to the GDF-15 threshold (897.00 pg / mL), into a negative group (less than or equal to the GDF-15 threshold) and a positive group (greater than the GDF-15 threshold). The GDF-15 negative group was the group with negative vascular plaque-related clinical events. Further, within the GDF-15 positive group, the NT-IGFBP-4 threshold (90.4 ng / mL) was used for further subdivision. The NT-IGFBP-4 positive group was the group with positive vascular plaque-related clinical events, while the NT-IGFBP-4 negative group was the observation group, which required confirmation based on other clinical indicators such as imaging.

[0092] The diagnostic efficacy of the combined application of NT-IGFBP-4 and GDF-15 in a diagnostic decision tree is shown in Table 4. The sensitivity and specificity were 84% and 51%, respectively, while the positive predictive value and negative predictive value were 91% and 36%, respectively. Compared to the individual diagnosis of the biomarkers, the combined diagnosis significantly improved diagnostic sensitivity and positive predictive value, enabling timely identification of the vast majority of individuals with actual vascular plaque risk. Furthermore, 91% of patients diagnosed positive by the combined biomarker diagnosis actually experienced vascular plaque-related clinical events, indicating that the positive results of the biomarkers have high predictive reliability and contribute to early disease identification and rapid triage.

[0093] Table 4. Thresholds and performance (decision tree) of the combined diagnostic model of NT-IGFBP-4 and GDF-15 for vascular plaque-related clinical events.

[0094] markers Cut-off Sensitivity Specificity Positive predictive value Negative predictive value NT-IGFBP-4+ GDF-15 GDF-15>897.00pg / mLNT-IGFBP-4>90.4ng / mL 84% 51% 91% 36%

[0095] The results of the above examples show that the method of using NT-IGFBP-4 and GDF-15 as biomarkers to diagnose vascular plaque-related clinical events is very simple and highly practical. Results can be obtained in a short time with only the following operation: First, collect blood samples from the subject, and obtain serum or plasma by standing and centrifugation; use specific immunoassay methods such as chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the concentration of NT-IGFBP-4 and GDF-15 in the sample. The diagnosis is then made. The NT-IGFBP-4 concentration can be compared individually with a reference threshold (e.g., 119.2 ng / mL), or the GDF-15 concentration can be compared with a reference threshold (e.g., 1221.00 pg / mL). A value above the threshold indicates a positive clinical event related to vascular plaque. Alternatively, a combined diagnosis can be performed by inputting the concentrations of the two biomarkers into a pre-defined combined diagnostic model (e.g., a binary logistic regression model: combined parameters = -15.982 + 5.694 × [NT-IGFBP-4] + 1.891 × [GDF-15]), or by classifying the results using a decision tree model (first determining GDF-15 > 897.00 pg / mL, then determining NT-IGFBP-4 > 90.4 ng / mL). The final diagnosis is given based on the probability or classification result output by the model. This process supports automated and standardized operation and can be combined with clinical information and other detection indicators for auxiliary diagnosis, early screening, and risk assessment.

[0096] It should be understood that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of N-terminal insulin-like growth factor binding protein-4 (NT-IGFBP-4) and / or growth differentiation factor-15 (GDF-15) as biomarkers in the preparation of diagnostic reagents for vascular plaque-related clinical events; or the use of NT-IGFBP-4 and / or GDF-15 as drug targets in the development of drugs for the treatment of vascular plaques.

2. The application or use as described in claim 1, characterized in that, The term "vascular plaque" refers to arterial plaque, especially coronary artery plaque.

3. The application or use as described in claim 1, characterized in that, The NT-IGFBP-4 level in the biological sample refers to the concentration / content of NT-IGFBP-4 protein, or the gene / mRNA expression level of NT-IGFBP-4; the GDF-15 level in the biological sample refers to the concentration / content of GDF-15 protein, or the gene / mRNA expression level of GDF-15.

4. The application as described in claim 1, characterized in that, The biological samples are selected from the following group: whole blood, plasma, serum, lung tissue samples, tissue fluid, saliva, oral mucosa, nasopharyngeal secretions, body fluids, and urine, with serum being the preferred biological sample.

5. The application or use as described in claim 1, characterized in that, When the level / concentration of NT-IGFBP-4 in a biological sample is more than 13.8% higher than the average level of healthy individuals, and the serum NT-IGFBP-4 level reaches more than 119.2 ng / mL; or / and the level / concentration of GDF-15 is more than 45% higher than the average level of healthy individuals, and the serum GDF-15 level reaches more than 1221 pg / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event; substances that inhibit the expression of NT-IGFBP-4 and / or GDF-15 are candidates for vascular plaque treatment, especially arterial plaque treatment drugs.

6. The application as described in claim 1, characterized in that, The reagent is a kit, specifically an NT-IGFBP-4 and / or GDF-15 detection kit for diagnosing vascular plaque-related clinical events, particularly arterial plaque endpoint events.

7. The application as described in claim 6, characterized in that, The kit is used to detect the concentration / content of NT-IGFBP-4 and / or GDF-15 proteins in biological samples, and can be a latex immunoturbidimetric assay kit, a magnetic microparticle chemiluminescence assay kit, a radioimmunoassay kit, or an immunochromatographic test strip.

8. The application as described in claim 6, characterized in that, The kit is a double-antibody sandwich assay kit, selected from the following group of methods: A. Enzyme-linked immunosorbent assay kit; B. Magnetic microparticle chemiluminescence assay kit; C. Immunochromatographic test strips.

9. The application as described in claim 6, characterized in that, The kit also includes a carrier containing mathematical models for diagnosing clinical events related to vascular plaques, the mathematical models being divided into three categories: First, the single biomarker NT-IGFBP-4 or GDF-15 is used for judgment. When the NT-IGFBP-4 level is more than 13.8% higher than the average level of healthy individuals and the serum NT-IGFBP-4 level reaches more than 119.2 ng / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event. Alternatively, when the GDF-15 level / concentration is more than 45% higher than the average level of healthy individuals and the serum GDF-15 level reaches more than 1221 pg / mL, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event. Second, the binary logistic regression model for joint / combined judgment of NT-IGFBP-4 and GDF-15 dual biomarkers is shown in the following formula: Joint inspection parameters = -15.982 + 5.694 × [NT-IGFBP-4] + 1.891 × [GDF-15] In the formula, [NT-IGFBP-4] is the serum detection concentration of NT-IGFBP-4, [GDF-15] is the serum detection concentration of GDF-15, and the cut-off value is 0.7204. The [NT-IGFBP-4] and [GDF-15] values ​​obtained from the detection are entered into the formula for calculation. When the calculated value is above 0.7204, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event. Thirdly, the decision tree model for the combined / integrated judgment of dual biomarkers NT-IGFBP-4 and GDF-15 has a serum detection concentration threshold (Cut-off) of firstly GDF-15 not less than 897.00 pg / mL and then NT-IGFBP-4 not less than 90.4 ng / mL. When the detected values ​​of [NT-IGFBP-4] and [GDF-15] are greater than this threshold, it is basically determined to be a vascular plaque-related clinical event, especially an arterial plaque endpoint event.

10. The use as described in claim 1, characterized in that, The vascular plaque treatment drug is an inhibitor of NT-IGFBP-4 and / or GDF-15 gene expression.

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