Biomarkers for atherosclerosis diagnosis and use thereof

Abstract

The application discloses biomarkers for atherosclerosis diagnosis and application thereof. The biomarkers for atherosclerosis diagnosis include any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4). The biomarkers provided by the application have a strong correlation with advanced atherosclerotic plaques, and can be used for diagnosis and experimental research of atherosclerosis, especially advanced atherosclerotic plaques.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to biomarkers for the diagnosis of atherosclerosis and their applications. Background Technology

[0002] Atherosclerosis (AS) is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Atherosclerosis is a chronic inflammation of the arterial wall, characterized by the gradual accumulation of lipids (such as cholesterol), cells (such as macrophages, T lymphocytes, or smooth muscle cells), and extracellular matrix. Large accumulations are called atherosclerosis or plaques, and they often contain calcium. Adipose tissue can erode the arterial wall, reducing its elasticity and affecting blood flow. Finally, a blood clot forms around the plaque accumulation, further obstructing blood flow and eventually completely blocking the blood vessel.

[0003] Atherosclerosis is the most common arterial disease and can further lead to thromboembolism. Atherosclerotic plaques often narrow the arterial lumen, causing local ischemia and sometimes tissue atrophy in the perfused area. Serious consequences include angina pectoris due to myocardial ischemia, heart failure due to ischemic or non-ischemic events, hypertension due to renal artery stenosis, and increased blood perfusion in response to increased renin secretion in the kidneys.

[0004] Complications or consequences of atherosclerosis include coronary artery disease (coronary atherosclerosis), insufficient blood supply due to blockage (local ischemia / angina), acute myocardial infarction (myocardial infarction, heart attack), transient ischemic attack (TIA) or stroke, and damage to blood vessels, muscles or body organs.

[0005] Depending on the stage of atherosclerosis development, atherosclerotic plaques can be classified into early, intermediate, and late stages. Late-stage plaques are extremely unstable and, upon detachment, can directly obstruct blood vessels, leading to serious consequences such as acute myocardial infarction, posing a significant threat. Therefore, the qualitative and spatial localization of unstable plaques, especially late-stage unstable plaques, is crucial for the identification and diagnosis of atherosclerosis.

[0006] Traditional methods for identifying and diagnosing atherosclerotic plaques often rely on HE staining, immunohistochemistry, and other techniques to determine plaque development by observing cell morphology and other characteristics. These methods usually depend on the researcher's personal experience and subjective understanding, making it difficult to quantify and standardize them.

[0007] In recent years, studies have used molecular imaging technology to visualize the progression of atherosclerotic inflammation by analyzing the pathological characteristics of unstable plaques.

[0008] For example, reference 1 describes the development of fluorescently labeled dual-ligand iron oxide nanoparticles (DT-MPIO) to counteract VCAM-1 and p-selectin. Adhesion molecules were detected and characterized by MRI in an apolipoprotein E-deficient (ApoE- / -) mouse model. In vivo MRI was used to assess the extent to which DT-MPIO induced adhesion molecules and inflammatory MR signal trajectories. The conclusion demonstrates that DT-MPIO can serve as a magnetic resonance imaging probe to characterize inflammation in atherosclerosis. Oxidation-specific epitopes (OSEs) play a crucial role in the initiation, progression, and instability of atherosclerotic plaques.

[0009] Reference 2 demonstrates that irbesartan's therapeutic effect on atherosclerosis can be achieved through the use of... 18 F-FDG and 99m Tc-annexin A5 was detected using molecular imaging. Reference 3 demonstrates that in CD80-specific tracers... 18 FFDG and 18 FFDM accumulates in atherosclerotic plaques. Reference 4 successfully prepared Fe-PFH (perfluorohexane phase change material)-polylactic acid-glycolic acid (PLGA) / chitosan (CS)-dextran sulfate (DS) nanoparticles (Fe-PFH-PLGA / CS-DS NPs) probes. These probes, combined with low-intensity focused ultrasound (LIFU) irradiation, enabled ultrasound imaging. Fe-PFH-PLGA / CS-DS NPs can be used as multi-peak and multifunctional probes, potentially enabling specific diagnosis and targeted therapy of vulnerable plaques.

[0010] Although the cited literature has studied individual molecules associated with unstable plaques in atherosclerosis using techniques such as MR molecular imaging, PET molecular imaging, or ultrasound molecular imaging, the exact pathogenesis of atherosclerosis at the molecular level is still not fully understood.

[0011] Therefore, further in-depth research is needed on the molecular pathogenesis of atherosclerosis, especially on the effective identification, diagnosis, and pathological study of late-stage unstable plaques.

[0012] References

[0013] Citation 1: Chan JMS, Monaco C, Wylezinska-Arridge M, et al. Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging[J]. J Vasc Surg, 2018, 67(5):1571-1583.

[0014] Citation 2: Zhao Y, Watanabe A, Zhao S, et al. Suppressive effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques of ApoE / mice:molecular imaging with 14 C-FDG and 99m Tc-annexin A5[J]. PLoS One, 2014, 9(2):89338-89340.

[0015] Citation 3: Meletta R, Steier L, Borel N, et al. CD80 is upregulated in a mouse model with shear stress-induced atherosclerosis and allows for evaluating CD80-targeting PET tracers[J]. Mol Imaging Biol, 2017, 19(1):90-99.

[0016] Citation 4: Ye M, Zhou J, Zhong Y, et al. SR-A-targeted phase-transition nanoparticles for the detection and treatment of atherosclerotic vulnerable plaques[J]. ACS Appl Mater Interfaces, 2019, 11(10):9702-9715. Summary of the Invention

[0017] The problem the invention aims to solve

[0018] Atherosclerosis is a major cause of coronary heart disease and ischemic stroke, a crucial pathological basis for cardiovascular and cerebrovascular diseases, and one of the major chronic diseases affecting human health. The typical manifestation of atherosclerosis is lipid accumulation in the arterial walls. Many studies have confirmed that atherosclerosis is an inflammatory lesion of the arterial wall, but its exact molecular pathogenesis is not yet fully understood. Based on its stage of development, atherosclerotic lesions can be classified into early, intermediate, and late-stage plaques. Late-stage plaques are extremely unstable; once they detach, they can directly obstruct blood vessels, leading to serious consequences such as acute myocardial infarction, posing a significant threat.

[0019] Traditional methods for identifying atherosclerotic plaques, such as HE staining and immunohistochemistry, rely on researchers' personal experience and subjective understanding, making it difficult to achieve indexing and standardization. Furthermore, although references 1-4 have investigated individual molecules, the exact pathogenesis of atherosclerosis at the molecular level is still not fully understood and requires further investigation.

[0020] This invention aims to address the challenges of directly categorizing and spatially locating unstable plaques in atherosclerosis research. Focusing on the aortic arch, a typical site of atherosclerosis in mouse samples, this invention employs micro-mass spectrometry to investigate the chemical composition and spatial distribution of unstable plaques in atherosclerotic model tissue within the aortic arch. This allows for the screening of spatially specific small-molecule biomarkers for unstable plaques.

[0021] Solution for solving the problem

[0022] After dedicated research, the inventor discovered that the above-mentioned technical problems can be solved through the following solution:

[0023] [1]. The present invention first provides a biomarker for the diagnosis of atherosclerosis, wherein the biomarker includes any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4).

[0024] [2]. According to the biomarkers described in [1], wherein the mass-to-charge ratios of the biomarkers are respectively:

[0025] PG(38:7): m / z 791.49; PG(34:2): m / z 745.50; PG(34:3): m / z 743.49; PG(38:6): m / z 793.50; PG(36:6): m / z 767.49; PG(38:5): m / z 795.52; PG(36:3): m / z 771.52; PG(36:4): m / z 769.50.

[0026] [3]. The biomarker according to [1] or [2], wherein the diagnosis is to determine the type of atherosclerotic plaque in a sample from the subject;

[0027] Preferably, the diagnosis is to determine whether the atherosclerotic plaques in the sample from the subject are late-stage plaques and / or unstable plaques.

[0028] [4]. The biomarker according to any one of [1] to [3], wherein the subject is a mammal.

[0029] [5]. The biomarker according to any one of [1] to [4], wherein the sample is atherosclerotic tissue.

[0030] [6]. The biomarker according to any one of [1] to [5], wherein the sample is atherosclerotic tissue pretreated by one or more of the following pretreatment methods: embedding, slicing and matrix coating.

[0031] [7]. Furthermore, the present invention also provides a kit for diagnosing atherosclerosis, wherein the kit contains a detection reagent for detecting biomarkers such as any one of [1] to [6] in a sample from a subject.

[0032] [8]. In addition, the present invention also provides a system for diagnosing atherosclerosis, wherein the system includes a detection device, a computing device and an output device;

[0033] The detection device includes a sampler and a detector, the sampler being used to collect samples from the subject, and the detector being used to detect biomarkers in the samples as described in any one of [1] to [6];

[0034] The computing device includes a memory and a processor. The memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to perform the following discrimination:

[0035] If the biomarker in the sample, as described in any one of [1] to [6], is highly expressed, then the atherosclerotic plaque of the subject corresponding to the sample is determined to be a late plaque and / or an unstable plaque.

[0036] [9]. According to the system described in [8], the detection device is an instrument with optical microscopy and mass spectrometry imaging functions.

[0037]

[10] . The system according to [9], wherein the sample is a slice of atherosclerotic tissue.

[0038]

[11] . The system according to any one of [8] to

[10] , wherein the output device is used to output the detection result of the detection device and / or the discrimination result of the computing device, and the output device includes at least one of a display, a printer and an audio output device;

[0039] The computing device includes at least one of a computer host, a central processing unit, and a network server.

[0040]

[12] . Use of any of the biomarkers described in any of [1] to [6] in the preparation of reagents or kits for diagnosing atherosclerosis in subjects.

[0041] The effects of the invention

[0042] By implementing the above technical solutions, the present invention can achieve the following technical effects:

[0043] This invention utilizes microscopic mass spectrometry imaging technology to conduct experimental research on a mouse aortic arch atherosclerosis model. Based on the experimental results, several potential markers with a strong correlation to late-stage atherosclerotic plaques were identified. Imaging mass spectrometry technology provides accurate material localization, qualitative and quantitative information for pathological research of atherosclerotic diseases at the molecular level, and is expected to provide more reliable experimental data and basic information for pathological research and applications in related diseases in the future. Attached Figure Description

[0044] Figure 1 This is a flowchart of the iMScope TRIO imaging mass spectrometry microscope and its imaging mass spectrometry workflow.

[0045] Figure 2 To confirm the location of late-stage plaques using optical micrographs.

[0046] Figure 3 Comparison of integrated mass spectra of early, middle and late plaque regions.

[0047] Figure 4 These are eight late-stage plaque-specific highly expressed biomarkers.

[0048] Figure 5 This is a schematic diagram showing the results of diagnosing human clinical vascular longitudinal section samples with atherosclerotic plaques using the biomarkers provided by this invention. Detailed Implementation

[0049] The present invention will now be described in detail. The descriptions of the technical features described below are based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

[0050] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0051] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0052] Unless otherwise specified, "%" in this instruction manual refers to the percentage of mass content.

[0053] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0054] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0055] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0056] In this specification, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0057] In this specification, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0058] This invention utilizes micro-mass spectrometry imaging technology to collect and selectively extract different spatially distributed biomarkers from atherosclerotic samples in the aortic arch of mice in a single sampling process, enabling the identification and diagnosis of advanced plaques at the molecular level. By leveraging micro-mass spectrometry imaging, this invention obtains a series of small molecule compounds, along with their varying levels and trends, that exhibit strong spatial distribution correlations with atherosclerotic lesions, particularly advanced plaques. These are then identified as targeted biomarkers for advanced plaques, allowing for precise qualitative and quantitative localization of these plaques. This provides fundamental reference indicators for research on the pathogenesis of this disease and for evaluating the efficacy of drug treatments.

[0059] The technical solution of the present invention will be described in detail below.

[0060] <Biomarkers for the diagnosis of atherosclerosis>

[0061] In a first aspect of the invention, a biomarker for the diagnosis of atherosclerosis is provided, the biomarker comprising any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3), and PG(36:4).

[0062] In this invention, a biomarker refers to a marker of disease, which is typically a readily measurable substance found in a subject's sample. Qualitative and quantitative measurements, as well as the determination of its location, can be related to the underlying disease pathophysiology, such as the presence of atherosclerosis, the type of atherosclerotic plaque, and the location of different types of plaque, or to its prognosis (i.e., the likelihood of overcoming the underlying disease). In patients receiving treatment for their condition, qualitative and quantitative measurements, as well as the determination of their location, may also be related to the response to treatment.

[0063] In this invention, the biomarker is one or more PG phospholipids (phosphatidylglycerols), preferably PG phospholipids whose fatty acid chains contain 30 to 40 carbon atoms, more preferably PG phospholipids whose fatty acid chains contain 32 to 40 carbon atoms, and even more preferably PG phospholipids whose fatty acid chains contain 34 to 38 carbon atoms, including any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4).

[0064] In this invention, PG(38:7) is used as an example, where "38" represents the total number of carbons in the fatty acid chains of the PG phospholipid molecule, and "7" represents the total number of double bonds in the fatty acid chains of the PG phospholipid molecule. Other listed PG phospholipids are represented in the same way.

[0065] In some preferred embodiments of the present invention, the biomarker includes any one or any combination of PG(36:6), PG(34:3), PG(38:5) and PG(34:2).

[0066] In this invention, atherosclerosis encompasses all diseases or conditions typically described as atherosclerotic, in which fatty substances accumulate in the vessel walls, eventually leading to narrowing and damage of blood flow, and the formation of thrombi causing rupture and / or erosion. Atherosclerosis includes the pathological conditions of atherosclerosis and its complications or sequelae, as described in detail in the "Background Art" section above.

[0067] In this invention, diagnosis includes the detection or identification of a subject's disease state or condition, determining the likelihood that a subject will have a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or its possible progression or regression) of a subject with a disease or condition, and determining the effect of treatment on a subject with a disease or condition. For example, diagnosis can be used to detect the presence or likelihood of a subject having atherosclerosis or the likelihood that such a subject will advantageously respond to a compound (e.g., a drug, pharmaceutical product) or other treatment.

[0068] In some specific embodiments of the invention, diagnosis also refers to determining the type of atherosclerotic plaque and locating different types of plaques, such as differentiating and locating early, intermediate, or late-stage plaques, or differentiating stable and unstable plaques. In some more specific embodiments of the invention, diagnosis is the characterization and location of late-stage and / or unstable plaques.

[0069] In some specific embodiments, the biomarkers provided by this invention can also be used to assist in determining the degree of atherosclerotic plaque development. In other specific embodiments, the biomarkers provided by this invention can also be used to assist in diagnosing the degree of atherosclerotic disease development.

[0070] In this invention, atherosclerotic plaques refer to the accumulation and expansion on the arterial wall, where the expansion is caused by, for example, macrophages, cellular debris, lipids such as cholesterol and phospholipids, calcium, and fibrous connective tissue.

[0071] Based on the harmfulness of atherosclerotic plaques, they can be classified into stable plaques and unstable plaques. Specifically, in this invention, plaques that are prone to thrombosis, have a tendency to rupture, or progress rapidly, thus posing a very high risk of stroke, are referred to as "vulnerable plaques," "unstable atherosclerotic plaques," or "unstable plaques." The nature of a plaque (stable or vulnerable) mainly depends on its composition; thin / ruptured fibrous caps, large lipid cores, and intraplaque hemorrhage are well-established characteristics of plaque vulnerability.

[0072] In this invention, "stable atherosclerotic plaque" or "stable plaque" refers to an atherosclerotic plaque in an individual that has not shown signs of development over a predetermined period of 6 months or longer.

[0073] Based on the development process, morphology, lipid and cellular components of atherosclerotic plaques, they can be classified into types I-VI. Types I and II are defined as initial plaques and fatty streaks, respectively, both representing early-stage plaques. Their main characteristics include an increase in intimal macrophages, the appearance of intracellular lipid droplets, the formation of a foamy macrophage layer, the presence of lipid-rich smooth muscle cells, granular substances within the plaque, and extracellular lipid droplets of various shapes. Type III plaques, also known as intermediate-stage plaques, represent the intermediate stage between early and late-stage plaques. In addition to possessing all the characteristics of type II plaques, their most significant feature is the continuous aggregation of extracellular lipid droplets, forming numerous small lipid pools. Types IV-VI plaques are collectively referred to as late-stage plaques, characterized by the formation of large lipid pools, the presence of fibrotic and calcified components, plaque cracking, and the appearance of hematomas or hemorrhages. Late-stage plaques often induce thrombosis, causing pathogenic or fatal medical events. In this invention, late-stage plaques are typically unstable, i.e., unstable plaques.

[0074] In some specific embodiments of the present invention, the mass-to-charge ratios of each biomarker (PG phospholipid) are as follows:

[0075] PG(38:7): m / z 791.49; PG(34:2): m / z 745.50; PG(34:3): m / z 743.49; PG(38:6): m / z 793.50; PG(36:6): m / z 767.49; PG(38:5): m / z 795.52; PG(36:3): m / z 771.52; PG(36:4): m / z 769.50. Here, the mass-to-charge ratio (m / z) refers to the ratio of the mass to the charge of the ionized molecule. The mass-to-charge ratio in this invention is accurate to two decimal places.

[0076] <Reagent kit for diagnosing atherosclerosis>

[0077] A second aspect of the invention provides a kit for diagnosing atherosclerosis, comprising detection reagents for detecting the biomarkers described in the first aspect of the invention in samples from subjects.

[0078] In the second aspect of the present invention, terms such as “biomarker,” “atherosclerosis,” “diagnosis,” and “atherosclerotic plaque” are the same as those in the first aspect of the present invention and have the same meaning as described in the first aspect.

[0079] In this invention, the subject may be a mammal or a cell, tissue, organ, or part of said mammal. In this invention, mammal means any kind of mammal, preferably a human (including a human subject or human patient). Subjects and mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats. In some preferred embodiments of this invention, the subject is a human, mouse, or rat.

[0080] In this invention, the detection reagent can be any reagent capable of qualitatively, quantitatively, and / or locally detecting the aforementioned biomarkers (PG phospholipids) in samples from a subject. In some specific embodiments of the invention, the detection reagent is suitable for mass spectrometry. In other specific embodiments of the invention, the detection reagent is suitable for magnetic resonance imaging (MRI), positron emission tomography (PET), or ultrasound molecular imaging. In some preferred embodiments, the detection reagent is suitable for imaging mass spectrometry microscopy. Imaging mass spectrometry microscopy enables the qualitative, quantitative, and / or locally identifiable detection of the aforementioned PG phospholipids in samples from a subject, thereby diagnosing atherosclerosis, particularly the presence and location of late-stage and / or unstable plaques. Furthermore, in this invention, when using the kit to detect / identify biomarkers, corresponding detection equipment is typically required, such as a mass spectrometer, magnetic resonance imaging instrument, PET instrument, ultrasound molecular imaging instrument, and / or imaging mass spectrometry microscope, preferably an imaging mass spectrometry microscope, such as the iMScope TRIO imaging mass spectrometry microscope.

[0081] In this invention, a sample refers to any substance that may contain target molecules that need to be analyzed, including biological samples. As used herein, a biological sample means any sample obtained from a live or viral (or prion) source or other macromolecular and biomolecular source, and includes any cell type or tissue of a subject from which nucleic acids, proteins and / or other macromolecules can be obtained. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow samples, ascites, pelvic lavage fluid, pleural fluid, cerebrospinal fluid, lymph, eye discharge, extracts from nasal, throat or genital swabs, cellular suspensions of digestive tissues, or extracts of fecal matter, and tissue and organ samples from humans, animals (e.g., non-human mammals) and plants, and processed samples derived therefrom.

[0082] In some specific embodiments of the invention, the sample or biological sample is atherosclerotic tissue, which can encompass any area or portion of a blood vessel exhibiting symptoms of atherosclerosis, including, for example, atheromas or atherosclerotic lesions / plaques. In some preferred embodiments of the invention, the atherosclerotic tissue is atherosclerotic tissue originating from the aortic arch.

[0083] In some specific embodiments of the present invention, the sample or biological sample may be pretreated according to the selected detection method before detection. Those skilled in the art can determine how to pretreat the sample according to the selected method. For example, when detecting by imaging mass spectrometry, atherosclerotic tissue may be embedded, sectioned (e.g., frozen section), and coated with matrix, for example by spraying or sublimation.

[0084] <Uses of Biomarkers in the Diagnosis of Atherosclerosis>

[0085] In a third aspect of the invention, the use of a biomarker selected from the following in the preparation of a reagent or kit for diagnosing atherosclerosis in a subject is provided: any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4).

[0086] A fourth aspect of the invention provides for the use of biomarkers selected from the following in atherosclerosis research: any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4).

[0087] In some preferred embodiments of the present invention, the biomarker includes any one or any combination of PG(36:6), PG(34:3), PG(38:5) and PG(34:2).

[0088] In this invention, the study of atherosclerosis includes the study of animal models of atherosclerosis, the pathological study of atherosclerosis, and the study of the pathogenesis of atherosclerosis at the molecular level.

[0089] Furthermore, in the third and fourth aspects of the present invention, terms such as "biomarker," "atherosclerosis," "diagnosis," and "atherosclerotic plaque," which are the same as those in the foregoing aspects of the present invention, have the same meanings as described in the foregoing aspects.

[0090] Diagnostic System for Atherosclerosis

[0091] A fifth aspect of the invention provides a system for diagnosing atherosclerosis, wherein the system includes a detection device, a computing device, and an output device;

[0092] The detection device includes a sampler and a detector. The sampler is used to collect a sample from the subject, and the detector is used to detect any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4) in the sample.

[0093] The computing device includes a memory and a processor. The memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to perform the following discrimination:

[0094] If any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3), and PG(36:4) in the sample is highly expressed, then the atherosclerotic plaque of the subject corresponding to the sample is determined to be a late plaque and / or an unstable plaque.

[0095] In this invention, high expression means that it can accurately correspond to the patch tissue in the microscopic optical photograph, and at the same time, its display brightness in the mass spectrometry imaging is significantly higher than that of the surrounding tissue and other patch tissues.

[0096] In some preferred embodiments of the present invention, any one or any combination of PG(36:6), PG(34:3), PG(38:5) and PG(34:2) in the sample is detected.

[0097] In a fifth aspect of the invention, the detection device can be an instrument with optical microscopy and mass spectrometry imaging capabilities, preferably an imaging mass spectrometry microscope, such as the iMScope TRIO imaging mass spectrometry microscope. The output device is used to output the detection results of the detection device and / or the discrimination results of the computing device, and the output device includes at least one of a display, a printer, and an audio output device. The computing device includes at least one of a computer host, a central processing unit, and a network server.

[0098] Furthermore, in the fifth aspect of the present invention, terms such as "biomarker," "atherosclerosis," "diagnosis," and "atherosclerotic plaque," which are the same as those in the foregoing aspects of the present invention, have the same meanings as described in the foregoing aspects.

[0099] Diagnostic methods for atherosclerosis

[0100] A sixth aspect of the present invention provides a diagnostic method for diagnosing atherosclerosis, comprising the following steps:

[0101] Provide samples from the subjects;

[0102] The sample is tested for any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3), and PG(36:4);

[0103] To determine whether the atherosclerotic plaques in the subjects' samples were late-stage plaques and / or unstable plaques.

[0104] In some preferred embodiments of the present invention, any one or any combination of PG(36:6), PG(34:3), PG(38:5) and PG(34:2) in the sample is detected.

[0105] Furthermore, in the sixth aspect of the present invention, terms such as "biomarker," "atherosclerosis," "diagnosis," and "atherosclerotic plaque," which are the same as those in the foregoing aspects of the present invention, have the same meanings as described in the foregoing aspects.

[0106] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0107] Example

[0108] Example 1: Construction of a mouse model of atherosclerosis

[0109] Inducing apoE by feeding with a high-fat diet - / - Elevated blood lipids and atherosclerosis were observed in mice (high-fat diet formulation: 78.6% rat and mouse breeding diet, 10% lard, 1.00% cholesterol, 10% egg yolk powder, 0.4% bile salts). ApoE was isolated after 32 weeks of high-fat diet feeding. - / -Mouse aortic arch tissue blocks were obtained by fixing in neutral formalin, dehydrating, and embedding in paraffin. 4μm thick tissue sections were cut and stained with H&E. Plaque formation was observed under a microscope, and plaques in different locations of the aortic arch were classified. Early plaques: Increased number of endothelial macrophages, intracellular lipid droplet vacuoles, formation of a foamy macrophage layer, presence of lipid-rich smooth muscle cells, granular material, and extracellular lipid droplets of various shapes within the plaque. Intermediate plaques: In addition to exhibiting the pathological morphology characteristics of early plaques, numerous small lipid pools appear extracellularly. Late plaques: Large lipid pools appear within the plaque, fibrosis and calcification are present, plaques crack or rupture, and hematomas or hemorrhages occur.

[0110] Example 2: Screening of small molecule biomarkers for space-specific unstable plaques (late-stage plaques)

[0111] 1. Sample Preparation and Instrumentation: Mouse aortic arch tissue samples from the atherosclerosis mouse model constructed in Example 1 were embedded in gelatin solution (100 mg / mL), frozen, and sectioned using a cryostat. Optical images of the prepared sections were first captured, followed by matrix sublimation using an iMLayer and analysis using an iMScope TRIO imaging mass spectrometer. Figure 1 As shown.

[0112] Matrix application conditions

[0113] Matrix coating mode Sublimation (iMLayer) Matrix sublimation thickness 9AA: 0.9μm

[0114] Imaging mass spectrometry analysis conditions

[0115] Collection range m / z 500-1000 Laser diameter 40μm Acquisition interval 100μm

[0116] 2. Confirmation of location using in-situ optical microscopy images: Open the in-situ optical microscopy images taken at the same time using Imaging MS Solution Postrun Analysis to confirm the location of late-stage patches. For example... Figure 2 As shown.

[0117] 3. Software Processing and Calculation: Imaging MS Solution Postrun Analysis was used to process the collected data. Direct comparisons were made of the composite mass spectra of early, middle, and late-stage plaque regions within the m / z 500-1000 range. Figure 3As shown, images of eight fragments were extracted directionally: m / z 791.49 PG (38:7), m / z 745.50 PG (34:2), m / z 743.49 PG (34:3), m / z 793.50 PG (38:6), m / z 767.49 PG (36:6), m / z 795.52 PG (38:5), m / z 771.52 PG (36:3), and m / z 769.50 PG (36:4), respectively, as biospatial markers of late-stage plaques. Figure 4 As shown.

[0118] 4. Sample Verification: By correlating with the late-stage plaques indicated by optical micrographs and combining the extracted PG phospholipids [m / z 791.49 PG(38:7), m / z 745.50 PG(34:2), m / z 743.49 PG(34:3), m / z 793.50 PG(38:6), m / z 767.49 PG(36:6), m / z 795.52 PG(38:5), m / z 771.52 PG(36:3), m / z 769.50 PG(36:4)], the location and nature of the late-stage plaques were determined by several or all of these eight markers.

[0119] Test case

[0120] Mass spectrometry imaging data was acquired from longitudinal sections of vascular tissue with atherosclerotic plaques in human clinical practice, following the conditions described in Example 2. Mass spectrometry images of ions with m / z 767.49 (PG(36:6)), 743.49 (PG(34:3)), 795.52 (PG(38:5)), and 745.50 (PG(34:2)) were extracted. Figure 5 As can be seen, the high-expression areas (brightness significantly higher than the surrounding areas) are late-stage unstable plaques, and optical photographs show that they have a clear tendency to detach.

[0121] The descriptions of the exemplary embodiments presented above are merely illustrative of the technical solutions of the present invention and are not intended to be exhaustive or to limit the invention to the precise forms described. Obviously, those skilled in the art can make many changes and variations based on the above teachings. The exemplary embodiments were chosen and described to explain the specific principles of the invention and its practical applications, thereby enabling other those skilled in the art to understand, implement, and utilize the various exemplary embodiments of the invention and their various alternatives and modifications. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

[0122] Industrial availability

[0123] The biomarkers for the diagnosis of atherosclerosis provided by this invention can be used for the clinical diagnosis of atherosclerosis, as well as related basic research.

Claims

1. The use of biomarkers in the preparation of reagents or kits for diagnosing atherosclerosis in subjects, wherein, The biomarkers include any one or any combination of PG(38:7), PG(34:2), PG(34:3), PG(38:6), PG(36:6), PG(38:5), PG(36:3) and PG(36:4); The diagnosis involves determining the type of atherosclerotic plaques in samples from the subject. The mass-to-charge ratios of the biomarkers are as follows: PG (38:7): m / z 791.49; PG (34:2): m / z 745.50; PG (34:3): m / z 743.49; PG (38:6): m / z 793.50; PG (36:6): m / z 767.49; PG (38:5): m / z 795.52; PG (36:3): m / z 771.52; PG (36:4): m / z 769.

50.

2. The use according to claim 1, wherein, The diagnosis is to determine whether the atherosclerotic plaques in the sample from the subject are late-stage plaques and / or unstable plaques.

3. The use according to claim 1, wherein, The subjects mentioned are mammals.

4. The use according to claim 1, wherein, The sample in question is atherosclerotic tissue.

5. The use according to claim 1, wherein, The sample is atherosclerotic tissue that has been pretreated by one or more of the following methods: embedding, slicing, and matrix coating.

Images