Exosome-derived biomarker for diagnosing arteriosclerosis

Abstract

The present invention relates to exosome-derived biomarkers for diagnosing arteriosclerosis. The biomarkers of the present invention exhibit specific changes in expression associated with arteriosclerosis, particularly during the early stages of the disease, and thus are highly effective for early diagnosis and prognosis of arteriosclerosis. Particularly, the 3D arteriosclerosis model of the present invention closely mimics the arterial condition of actual patients, allowing for the combined use of the biomarkers and the model to enable early diagnosis, personalized treatment development, and precise monitoring of disease prognosis.

Description

Exosome-derived biomarkers for diagnosing atherosclerosis

[0001] The present invention relates to exosome-derived biomarkers for diagnosing arteriosclerosis.

[0002] Recently, along with the increase in adult diseases, cardiovascular diseases such as arteriosclerosis, hyperlipidemia, and myocardial infarction have been significantly increasing. Atherosclerosis, a representative cardiovascular disease, is a disease in which the arteries harden due to genetic factors related to lipid metabolism and environmental factors such as diet, smoking, and lack of exercise. It causes abnormalities in blood circulation in vital organs such as the brain, heart, and kidneys, and is a cause of cardiovascular diseases such as myocardial infarction, cerebral infarction, peripheral vascular disease, and aneurysm. It is easy for it to occur in the cerebral or coronary arteries, leading to cardiovascular diseases such as heart disease and cerebrovascular disease. Cerebral arteriosclerosis causes headaches, dizziness, and mental disorders, and can cause encephalomalacia, while coronary artery sclerosis causes pain and arrhythmia in the heart, which is known to cause angina and myocardial infarction. In addition, it causes high blood pressure, heart disease, and cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the biggest cause of death in modern society, especially for men in their 50s and 60s. It is known that arteriosclerosis is caused by damage to arterial blood vessels and cholesterol accumulation due to lipoproteins. The arterial wall is composed of the intima, which is a thin single layer of cells, the middle layer of the arterial wall, which is made of smooth muscle cells, and the adventitia, which plays a protective role in the artery. Atherosclerosis is caused by damage and degeneration of the arterial wall, which induces the formation of arteriosclerosis, but high blood pressure, hyperlipidemia, and smoking are important factors. In cases of hyperlipidemia, in which the cholesterol level in the blood is high, the phagocytes between the intima and the muscular layer of the artery contain a lot of fatty substances in their cytoplasm, causing fat accumulation in the arterial wall.

[0003] Plaque formation and rupture of blood vessels are major factors in the development of myocardial infarction, and can lead to serious complications of cardiovascular disease by impeding or blocking blood flow in the arteries. The initial onset of atherosclerosis is hypothesized to be a chronic inflammatory process caused by damage to the blood vessel wall. This is a state in which vascular endothelial cells become dysfunctional and unable to maintain normal homeostasis due to factors such as genetic mutations, peroxidation, hypertension, diabetes, increased plasma homocysteine ​​concentration, and / or microbial infection. Specifically, due to the above factors, low-density lipoprotein (LDL) in the blood undergoes oxidation, glycoconjugation, aggregation, and glycoprotein conjugation to transform into highly modified LDL (HM-LDL), which stimulates and damages vascular endothelial cells and smooth muscles. As a result, the expression of vascular cell adhesion molecule-1 (VCAM-1) of endothelial cells and the release of inflammatory mediators from inflammatory cells are promoted, causing LDL to infiltrate and accumulate under the endothelial cells, and the accumulated LDL and oxidized HM-LDL induce the influx and activation of immune cells such as macrophages and T lymphocytes, repeating the process to promote the inflammatory response of the lesion. The lesion becomes necrotic due to the action of hydrolytic enzymes, inflammatory mediators, and growth factors released from the infiltrated macrophages or lymphocytes, and a repetitive process such as the influx of monocytes into the necrotic lesion, the migration and differentiation of smooth muscle, and the formation of fibrous tissue occurs. Through the above process, the lesion tissue develops into a complex fibrous lesion with a fiber-covered necrotic tissue with HM-LDL as the core, and blood clots are formed from the developed lesion tissue, the muscle layer of the arterial wall becomes thicker, and the blood vessels become narrower, which can cause damage to various organs due to cardiovascular disease.

[0004] Atherosclerosis, also known as atherosclerosis or hardening of the arteries, is a vascular disease in which cholesterol is deposited in the endothelium, the innermost lining of the blood vessels, and endothelial cells proliferate, forming an "atheroma." When the inside of the blood vessel is blocked by an atheroma, blood circulation is impaired, leading to coronary artery disease or ischemic cerebrovascular disease. The main risk factors that cause the development and progression of atherosclerosis include high total cholesterol in the blood, low high-density lipoprotein cholesterol (HDL-cholesterol), high low-density lipoprotein cholesterol (LDL-cholesterol), high triglycerides, high blood pressure (140 / 90 mmHg or higher), smoking, diabetes, a family history of cardiovascular disease, increasing age, lack of exercise, overweight, and abdominal obesity. Atherosclerosis is diagnosed using imaging tests such as carotid ultrasound, computed tomography (CT) angiography (CTA), magnetic resonance angiography (MRA), cerebral angiography, and coronary angiography. While these imaging tests can identify the presence of arteriosclerosis in each patient, they are complex and prone to side effects and complications.

[0005] The purpose of the present invention is to provide a biomarker composition for diagnosing arteriosclerosis.

[0006] In addition, an object of the present invention is to provide a composition for diagnosing arteriosclerosis.

[0007] In addition, an object of the present invention is to provide a kit for diagnosing arteriosclerosis.

[0008] In addition, it is an object of the present invention to provide a method for providing information for diagnosing arteriosclerosis.

[0009] To solve the above problem, the present invention provides a biomarker composition for diagnosing arteriosclerosis.

[0010] In addition, the present invention provides a composition for diagnosing arteriosclerosis.

[0011] In addition, the present invention provides a kit for diagnosing arteriosclerosis.

[0012] In addition, the present invention provides a method for providing information for diagnosing arteriosclerosis.

[0013] The biomarkers of the present invention have expression changes specifically in arteriosclerosis, and are particularly specific in the early stage of the onset of arteriosclerosis, and thus are effective in being utilized for early diagnosis and prognosis prediction of arteriosclerosis. In particular, the 3D arteriosclerosis model of the present invention is more similar to the arterial condition of an actual patient, and thus, when used together, it is effective in being able to diagnose arteriosclerosis early, develop personalized treatment, and precisely monitor prognosis.

[0014] Figure 1 is a diagram showing exosomes secreted from cells of a 3D arteriosclerosis model of the present invention into tissues and exosomes secreted and present in tissues, confirmed by TEM:

[0015] Baseline: Before the onset of atherosclerosis;

[0016] Eearly athero: early stage of arteriosclerosis; and

[0017] Late athero: Late stage of arteriosclerosis.

[0018] Figure 2 is a diagram confirming the expression of positive exosome markers in the 3D arteriosclerosis model of the present invention using immunofluorescence staining:

[0019] Baseline: Before the onset of atherosclerosis;

[0020] Eearly athero: early stage of arteriosclerosis; and

[0021] Late athero: Late stage of arteriosclerosis.

[0022] Figure 3 is a diagram showing the results of nanoparticle tracking analysis of exosomes isolated from the 3D arteriosclerosis model of the present invention:

[0023] BL: Baseline;

[0024] EA: Eearly athero; and

[0025] LA: Late athero.

[0026] Figure 4 is a diagram showing quantitative analysis of the number of exosomes isolated from the 3D arteriosclerosis model of the present invention:

[0027] Baseline: Before the onset of atherosclerosis;

[0028] Eearly athero: early stage of arteriosclerosis; and

[0029] Late athero: Late stage of arteriosclerosis.

[0030] Figure 5 is a diagram showing the expression of exosome positive and negative markers in exosomes isolated from the 3D arteriosclerosis model of the present invention, analyzed by Western blot:

[0031] Baseline: Before the onset of atherosclerosis;

[0032] Eearly athero: early stage of arteriosclerosis; and

[0033] Late athero: Late stage of arteriosclerosis.

[0034] Figure 6 is a diagram confirming the expression of CD63 and CD9 in exosomes isolated from the 3D arteriosclerosis model of the present invention using flow cytometry:

[0035] Baseline: Before the onset of atherosclerosis;

[0036] Eearly athero: early stage of arteriosclerosis; and

[0037] Late athero: Late stage of arteriosclerosis.

[0038] Figure 7 is a diagram showing the expression of previously known cardiovascular disease biomarkers in exosomes isolated from the 3D arteriosclerosis model of the present invention and comparing them between each group:

[0039] BL: Before the onset of arteriosclerosis;

[0040] EA: Early atherosclerosis; and

[0041] LA: Late stage of atherosclerosis.

[0042] Figure 8 is a diagram showing the expression of exosome-derived protein biomarkers related to cardiovascular disease in exosomes isolated from the 3D arteriosclerosis model of the present invention and comparing them between each group:

[0043] BL: Before the onset of arteriosclerosis;

[0044] EA: Early atherosclerosis; and

[0045] LA: Late stage of atherosclerosis.

[0046] Figure 9 is a Venn diagram showing the protein expression patterns that change in the early and late atherosclerosis groups compared to the pre-atherosclerosis group in the 3D atherosclerosis model of the present invention:

[0047] Baseline: Before the onset of atherosclerosis;

[0048] Eearly athero: early stage of arteriosclerosis; and

[0049] Late athero: Late stage of arteriosclerosis.

[0050] Figure 10 is a heat map showing the expression of proteins that change over time during the development of arteriosclerosis:

[0051] BL: Before the onset of arteriosclerosis;

[0052] EA: Early atherosclerosis; and

[0053] LA: Late stage of atherosclerosis.

[0054] Figure 11 is a diagram illustrating previously unknown early atherosclerosis-specific protein biomarkers whose expression was more than doubled in the early atherosclerosis group:

[0055] BL: Before the onset of arteriosclerosis;

[0056] EA: Early atherosclerosis; and

[0057] LA: Late stage of atherosclerosis.

[0058] Figure 12 is a diagram showing markers highly correlated with cardiovascular disease derived from transcriptome big data analysis of the 14 biomarkers of Figure 11:

[0059] BL: Before the onset of arteriosclerosis;

[0060] EA: Early atherosclerosis; and

[0061] LA: Late stage of atherosclerosis.

[0062] Figure 13 is a diagram analyzing the interaction between the biomarker SERPINA11, whose expression increases in exosomes in the early stages of atherosclerosis, and other proteins.

[0063] Figure 14 is a diagram confirming the change in expression of F2 and AHSG proteins analyzed to interact with SERPINA11 in the 3D arteriosclerosis model of the present invention:

[0064] BL: Before the onset of arteriosclerosis;

[0065] EA: Early atherosclerosis; and

[0066] LA: Late stage of atherosclerosis.

[0067] Figure 15 is a diagram showing exosome-derived proteins whose expression increased by more than two-fold in the early and late atherosclerosis groups:

[0068] BL: Before the onset of arteriosclerosis;

[0069] EA: Early atherosclerosis; and

[0070] LA: Late stage of atherosclerosis.

[0071] Figure 16 is a diagram showing the biomarker of Figure 15 analyzed using transcriptome big data to derive the marker RANBP1, which is highly correlated with cardiovascular disease:

[0072] BL: Before the onset of arteriosclerosis;

[0073] EA: Early atherosclerosis; and

[0074] LA: Late stage of atherosclerosis.

[0075] Figure 17 is a diagram illustrating previously unknown late atherosclerosis-specific exosome-derived protein biomarkers whose expression was more than doubled in the late atherosclerosis group:

[0076] BL: Before the onset of arteriosclerosis;

[0077] EA: Early atherosclerosis; and

[0078] LA: Late stage of atherosclerosis.

[0079] Figure 18 is a diagram showing the biomarkers of Figure 17 analyzed through transcriptome big data to derive exosome-derived protein markers MAN1A1, TKFC, and UROC1, which are highly associated with cardiovascular disease:

[0080] BL: Before the onset of arteriosclerosis;

[0081] EA: Early atherosclerosis; and

[0082] LA: Late stage of atherosclerosis.

[0083] Figure 19 is a diagram showing microRNAs whose expression was reduced in the early atherosclerosis group among microRNA biomarkers that interact with the early atherosclerosis-specific protein biomarkers SERPINA11, F2, and AHSG:

[0084] BL: Before the onset of arteriosclerosis;

[0085] EA: Early atherosclerosis; and

[0086] LA: Late stage of atherosclerosis.

[0087] Figure 20 is a diagram showing the microRNAs targeting SERPINA11 and F2 proteins among the microRNAs of Figure 19:

[0088] BL: Before the onset of arteriosclerosis;

[0089] EA: Early atherosclerosis; and

[0090] LA: Late stage of atherosclerosis.

[0091] Figure 21 is a diagram analyzing miRNAs whose expression increases in the early group of atherosclerosis:

[0092] BL: Before the onset of arteriosclerosis;

[0093] EA: Early atherosclerosis; and

[0094] LA: Late stage of atherosclerosis.

[0095] Figure 22 is a diagram showing the microRNA targeting AHSG among the miRNAs of Figure 21:

[0096] BL: Before the onset of arteriosclerosis;

[0097] EA: Early atherosclerosis; and

[0098] LA: Late stage of atherosclerosis.

[0099] Figure 23 is a diagram showing changes in the expression of biomarkers for early diagnosis of arteriosclerosis derived from the present invention:

[0100] BL: Before the onset of arteriosclerosis;

[0101] EA: Early atherosclerosis; and

[0102] LA: Late stage of atherosclerosis.

[0103] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0104] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0105] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0106]

[0107] In one aspect, the present invention relates to a biomarker composition for diagnosing arteriosclerosis, comprising at least one selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a and miR-6879-5p.

[0108] In one embodiment, the atherosclerosis may occur in the aorta, coronary artery, basilar artery, renal artery, or peripheral artery.

[0109] In one embodiment, the arteriosclerosis may be inflammatory coronary arteriosclerosis.

[0110] In one embodiment, the biomarker may be expressed in blood or plasma-derived exosomes at the onset of atherosclerosis.

[0111] In one embodiment, RANBP1 may have altered expression specifically in early and late atherosclerosis, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a and miR-6879-5p may have altered expression specifically in early atherosclerosis, and MAN1A1, TKFC and UROC1 may have altered expression specifically in late atherosclerosis.

[0112] In one embodiment, RANBP1 may have increased expression in early and late atherosclerosis, SERPINA11, F2, hsa-miR-6879-5p and hsa-miR-1260a may have increased expression in early atherosclerosis, and MAN1A1, TKFC and UROC1 may have increased expression in late atherosclerosis.

[0113] In one embodiment, AHSG and hsa-miR-143-5p may be reduced in expression in early atherosclerosis.

[0114] In one embodiment, the biomarker composition may include at least one selected from the group consisting of SERPINA11 (serpin A11), F2 (prothrombin), AHSG (Association of low fetuin-A), hsa-miR-143-5p, hsa-miR-1260a, and hsa-miR-6879-5p, and may include SERPINA11, F2, AHSG, hsa-miR-143-5p, hsa-miR-1260a, and hsa-miR-6879-5p, and the biomarker may be used for early diagnosis of arteriosclerosis.

[0115] In one embodiment, the expression of SERPINA11, F2, miR-6879-5p and miR-1260a may be increased by the development of atherosclerosis.

[0116] In one embodiment, the expression of AHSG and miR-143-5p may be decreased by the development of atherosclerosis.

[0117] As used herein, the term "diagnosis" includes determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, determining the prognosis of an object suffering from a particular disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information about the efficacy of a treatment).

[0118] In one aspect, the present invention relates to a composition for diagnosing arteriosclerosis, comprising a formulation for measuring the expression level of one or more biomarkers selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a and miR-6879-5p.

[0119] In one embodiment, the agent for measuring the expression level of protein biomarkers RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2 and AHSG may be a peptide, antibody or immunologically active fragment thereof, aptamer, avidity multimer or peptidomimetics that specifically binds to the full-length protein or a fragment thereof, and the expression level may be measured by one or more methods selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry or protein microarray.

[0120] In one embodiment, the agent for measuring the expression level of the microRNA biomarkers miR-143-5p, miR-1260a and miR-6879-5p may be a peptide nucleic acid (PNA), an aptamer, a primer pair, a probe, or a primer pair and a probe that specifically recognize a nucleic acid sequence of the microRNA, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and the complementary sequence, and may be expressed by one or more methods selected from the group consisting of polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase protection assay (RPA), next generation sequencing (NGS), in situ hybridization, nucleic acid microarray, northern blot, DNA chip, multiplex PCR or ddPCR. The level can be measured.

[0121] In one embodiment, the immunologically active fragment of the antibody of the present invention may be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody.

[0122] The term “detection” or “measurement” used in the present invention means quantifying the concentration of a detected or measured object.

[0123] The term "primer" as used herein refers to a short nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

[0124] In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which is short, a few bases, or a long, several hundred bases, and can specifically bind to mRNA, and is labeled so that the presence or absence of a specific microRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the microRNA, and the level of microRNA expression can be diagnosed based on the presence or absence of hybridization. The selection of an appropriate probe and hybridization conditions can be modified based on those known in the art, and therefore the present invention does not specifically limit them.

[0125] The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0126] In the present invention, suitable conditions for hybridizing a probe with a microRNA molecule can be determined through a series of optimization procedures. These procedures can be implemented as a series of procedures by those skilled in the art to establish a protocol for use in a laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength depend on various factors such as the length and GC content of the probe and the target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., NY (1999). For example, among the above stringency conditions, high stringency conditions mean hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), 1 mM EDTA, and washing at 68°C in 0.1 x SSC (standard saline citrate) / 0.1% SDS. Alternatively, high stringency conditions mean washing at 48°C in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean washing at 42°C in 0.2 x SSC / 0.1% SDS, for example.

[0127] As used herein, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to the marker protein of the present invention, and the method for producing the antibody can be produced using a well-known method. This also includes a partial peptide that can be produced from the protein. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof that has antigen binding property is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

[0128] In one embodiment, a label may be additionally included, and the label may be a fluorescent substance, a probe, or a tag. The label may be any chemical moiety that can be detected by any method known in the art. Examples of detectable labels may include any moiety that can be detected by spectroscopy, photochemistry, biochemical, immunochemical, or chemical means. An appropriate method for labeling a nucleic acid probe may be selected depending on the type and location of the label and the probe. Examples of labels may include enzymes, enzyme substrates, isotopes, fluorescent dyes, chromophores, chemiluminescent labels, electrochemical luminescent labels, ligands having specific binding partners, and other labels that can react with each other to increase, modify, or decrease the intensity of a detection signal.

[0129] In one embodiment, the labeling agent may be a fluorescent agent, and the fluorescent agent may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), or modified red fluorescent protein (mRFP).

[0130] In one aspect, the present invention relates to a kit for diagnosing arteriosclerosis comprising the composition of the present invention.

[0131] In one embodiment, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

[0132] In one embodiment, the kit may further include tools and / or reagents for collecting a biological sample from a subject or patient, as well as tools and / or reagents for preparing genomic DNA, cDNA, RNA, or protein from the sample. For example, the kit may include PCR primers for amplifying relevant regions of genomic DNA. The kit may include probes for genetic factors useful for pharmacogenomic profiling. Furthermore, when using such a kit, labeled oligonucleotides can be used for easy identification during analysis.

[0133] In the present invention, the term "composition for diagnosing arteriosclerosis" refers to a substance that can predict / diagnose the risk of developing arteriosclerosis or diagnose arteriosclerosis at a specific stage, and includes organic biomolecules such as polypeptides or nucleic acids (e.g., miRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in expression with a significant correlation with the possibility of developing arteriosclerosis. For the purpose of the present invention, the biomarker for diagnosing arteriosclerosis is a marker whose miRNA expression or protein expression level specifically increases or decreases depending on the development or stage (early / late) of arteriosclerosis. These markers include not only the protein or nucleic acid of the marker itself but also a nucleic acid complementary to any one of the markers.

[0134] In one aspect, the present invention relates to a method for providing information for the diagnosis of atherosclerosis, comprising the step of measuring the expression level of one or more selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a and miR-6879-5p in a biological sample isolated from an individual.

[0135] In one embodiment, the biological sample may be blood or plasma derived exosomes.

[0136] The term "subject" or "individual" as used in the present invention means any animal, including a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, which has developed or may develop arteriosclerosis.

[0137]

[0138] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0139]

[0140] Example 1. Creation of a 3D arteriosclerosis model

[0141] 3D coronary artery tissue fabrication

[0142] Inflammatory macrophages (1x10) in ColMA hydrogel 6 cells / mL) (M1 macrophages), coronary artery smooth muscle cells (5×10 6 cells / mL) and coronary artery endothelial cells (2 × 10 6 cells / mL) were mixed to prepare bioink, and 3D printed in the order of coronary artery smooth muscle cells, inflammatory macrophages, and coronary artery endothelial cells using a bioprinting machine equipped with a syringe holder, UV LED, syringe, and bed temperature control device, and its software, NewCreatorK (Rokit, Seoul, South Korea). As a result, a homogeneous sheet structure (average diameter of approximately 7 mm and height of approximately 1.05 mm) with a porous, lattice-like meshwork structure having a structure of endothelial cells (0.15 mm thick) in the topmost layer, inflammatory macrophages (0.6 mm thick) in the middle layer, and smooth muscle cells (0.3 mm thick) in the bottommost layer was fabricated. The fabricated structure was irradiated with UV LED to crosslink the hydrogel, and then cultured in a 12-well plate to fabricate a 3D coronary artery tissue with a meshwork structure. Afterwards, 2 mg / mL of LDL was treated to the 3D coronary artery tissue to induce inflammatory arteriosclerosis, and the progression characteristics of arteriosclerosis were classified into the early athero group (day 0) before induction of arteriosclerosis, the time of onset, and the late athero group (day 1).

[0143]

[0144] Example 2. Analysis of exosomes secreted from a 3D atherosclerosis model.

[0145] The characteristics of exosomes (30-150 nm) secreted according to the progression stage of arteriosclerosis in the 3D arteriosclerosis model of Example 1 above were confirmed using transmission electron microscopy and immunofluorescence staining.

[0146] Transmission electron microscopy observed cells within 3D coronary artery tissues, and exosomes secreted from cells into tissues and exosomes remaining within tissues were observed in all groups: pre-atherosclerosis (BL; Baseline; Day 0), early atherosclerosis (EA; Early Athero; Day 3), and late atherosclerosis (LA; Late Athero; Day 14) (Fig. 1). In addition, immunofluorescence staining using antibodies against exosome markers CD9, CD63, and CD81 revealed that CD63-positive exosomes were observed within cells and tissues in all groups: pre-atherosclerosis, early atherosclerosis, and late atherosclerosis (Fig. 2).

[0147]

[0148] Example 3. Analysis of exosomes isolated from a 3D atherosclerosis model.

[0149] To analyze the characteristics of exosomes before confirming whether the composition of proteins and miRNAs secreted in each group changes according to the stage of arteriosclerosis, exosomes were purified from each group and nanoparticle tracking analysis, Western blot, and flow cytometry were performed.

[0150] Nanoparticle tracking analysis results showed that exosomes measuring 30-150 nm in size were secreted in large quantities in all groups (Fig. 3), and in particular, exosome secretion was significantly increased in the early and late atherosclerosis groups compared to the pre-atherosclerosis group (Fig. 4). In addition, Western blot analysis of exosome positive markers (CD9, CD63, and CD81) and negative markers (Calnexin) showed that positive markers (CD9, CD63, and CD81) were expressed in all groups, while negative markers (Calnexin) were not expressed (Fig. 5). In addition, flow cytometry analysis results showed that CD63 and CD9 positive exosomes were identified in the three groups (Fig. 6). Through this, it was confirmed that exosomes at each stage of atherosclerosis in the 3D atherosclerosis model of the present invention were purely isolated.

[0151]

[0152] Example 4. Analysis of expression of conventional cardiovascular disease biomarkers in a 3D atherosclerosis model.

[0153] In order to verify that the 3D arteriosclerosis model of the present invention simulates cardiovascular disease, exosomes secreted from the arteriosclerosis model were dissolved to isolate proteins, and then proteins expressed in the exosomes of each group were identified using a proteomics method, and among these, the expression levels of previously known cardiovascular disease biomarker proteins were analyzed.

[0154] As a result, acute phase reactants, CRP and SAA, increased in the early atherosclerosis group and the late atherosclerosis group compared to the baseline group, and increased in the early atherosclerosis group compared to the late atherosclerosis group (Fig. 7). In contrast, the expression of PAPPA, a marker related to plaque rupture, was found to increase in the late atherosclerosis group compared to the early atherosclerosis group (Fig. 7). In addition, it was confirmed that exosome-derived protein biomarkers associated with conventional cardiovascular diseases, such as APOD, APOC3, C1QA, C5, CRP, PIGR, SEPRING1, SERPINC1, and SERPINF2, were expressed in the 3D atherosclerosis model of the present invention (Fig. 8). Through this, it was confirmed that the protein expression changes according to time during the atherosclerosis induction process in the 3D atherosclerosis model of the present invention were similar to the expression patterns of conventional cardiovascular disease tissue and exosome-derived biomarkers, and therefore it can be seen that the new markers to be discovered in the 3D atherosclerosis model of the present invention can specifically diagnose and detect atherosclerosis.

[0155]

[0156] Example 5. Discovery of Exosome-Derived Early Atherosclerosis-Specific Protein Biomarkers

[0157] We analyzed proteins and miRNAs whose expression changed in exosomes secreted at different stages of atherosclerosis using proteomics and miRNA sequencing to discover novel biomarkers specific to early atherosclerosis. The results were plotted using a Venn diagram to identify exosome-derived proteins whose expression changed in the early and late atherosclerosis groups compared to the pre-atherosclerosis group (Fig. 9). Heat map analysis was used to visually analyze proteins whose expression changed over time during the atherosclerosis process (Fig. 10). Among the candidate biomarkers of early atherosclerosis-related proteins analyzed in this way, we analyzed proteins whose expression increased by more than twofold in the early atherosclerosis group, although they were not previously known. As a result, protein biomarkers of IGHV3-53, IGHV3-43D, IGHV2-5, IGKV1-16, ASCC3, IGHV1-3, IGHV3-20, SERPINA11, TPD52L2, IGKV1D-16, GRPEL1, MINPP1, IGHV3OR16-12, and IGHV3-35 were discovered (Fig. 11), and to determine potential atherosclerosis biomarker candidates among the above 14 proteins, transcriptome big data analysis was performed using the ARCHS4 data base. As a result of transcriptome big data analysis, among the above 14 proteins, ASCC3, SERPINA11, TPD52L2, GRPEL1, and MINPP1 were analyzed to be associated with cardiovascular disease, and among them, SERPINA11 was found to be the most highly associated with cardiovascular disease (Fig. 12), and was selected as an early atherosclerosis biomarker. To analyze the interaction between the SERPINA11 protein, an early atherosclerosis marker selected in this way, and other proteins, STRING was constructed using an existing database (Szklarczyk et al. Nucleic Acids Res. 2015 43(Database issue):D447-52).As a result, SERPINA11 formed a network with CEP350, EIF2B3, SERPINA7, F2, ALB, AHSG, LRRC27, LRRC66, PPIB, and ALG1L proteins (Fig. 13). Among the SERPINA7, F2, ALB, and AHSG proteins related to cardiovascular disease, the expression of the F2 protein was found to increase in the early and late atherosclerosis groups compared to the pre-atherosclerosis group, and the expression of the AHSG protein was found to decrease in the early and late atherosclerosis groups compared to the pre-atherosclerosis group (Fig. 14).

[0158]

[0159] Example 6. Discovery of Exosome-Derived Early and Late Atherosclerosis-Specific Protein Biomarkers

[0160] To identify novel atherosclerosis-related biomarker candidates whose expression persists from early to late atherosclerosis, we analyzed previously unknown exosome-derived proteins whose expression increased more than twofold in the early and late atherosclerosis groups. As a result, APOC3, CFI, APCS, APOA4, CD5L, BPGM, C1QA, HBE1, RANBP1, C4B, SPP1, SELL, CPN1, PKLR, MTPN, PNP, COPS8, PLXDC2, ANTXR2, PGLS, MYLK, APOE, F11, GCLC, FMOD, CAB39, MGAT1, and VTN proteins were identified as candidates (Fig. 15), and as a result of transcriptome big data analysis using the ARCHS4 data base, RANBP1, a protein associated with cardiovascular disease, was identified, and this was selected as a new atherosclerosis-related biomarker whose expression continuously changed from the early to late stages of atherosclerosis (Fig. 16).

[0161]

[0162] Example 7. Discovery of exosome-derived late-stage atherosclerosis-specific protein biomarkers.

[0163] To identify novel candidates for late-onset protein biomarkers associated with atherosclerosis, we analyzed previously unknown exosome-derived proteins whose expression was more than twofold increased in the late-onset atherosclerosis group. As a result, KIF3C, IGKV2-24, NMRAL1, IGLV2-14, MAN1A1, HOOK3, NEK9, RELCH, CLEC11A, KAZALD1, OLFML3, TKFC, PSMA3, DARS1, FAHD2A, UROC1, OR5A2, PCYOX1L, PSMA5, SEPTIN11, and ANKRD13A proteins were derived as candidates (Fig. 17), and as a result of transcriptome big data analysis through the ARCHS4 data base, KIF3C, IGKV2-24, NMRAL1, IGLV2-14, MAN1A1, HOOK3, NEK9, KAZALD1, TKFC, FAHD2A, UROC1, PCYOX1L, and PSMA5, ANKRD13A were derived as proteins associated with cardiovascular disease, and among these, the most highly correlated MAN1A1, TKFC, and UROC1 proteins were selected as novel late atherosclerosis biomarkers (Fig. 18).

[0164]

[0165] Example 8. Discovery of exosome-derived early atherosclerosis-specific miRNA biomarkers.

[0166] Since it is desirable to diagnose arteriosclerosis early to prevent cardiovascular disease, microRNA biomarkers that interact with the early arteriosclerosis-specific biomarkers SERPINA11, F2, and AHSG derived from Example 5 were discovered for early diagnosis of arteriosclerosis. As a result of analyzing miRNAs whose expression decreased in the early arteriosclerosis group, 37 miRNAs were found to have decreased expression (Fig. 19). As a result of analyzing miRNAs targeting SERPINA11 and F2 proteins among the 37 miRNAs using miRWalk analysis, hsa-miR-143-5p was found to target SERPINA11, and was selected as a marker, and no miRNA targeting F2 was derived (Fig. 20). In addition, as a result of analyzing miRNAs whose expression increased in the initial group of the arteriosclerosis model of the present invention, 114 miRNAs were derived (Fig. 21), and among them, miRNAs targeting AHSG were hsa-miR-6879-5p, hsa-miR-1260a, hsa-miR-4474-3p, hsa-miR-4728-3p, hsa-miR-6731-3p, and hsa-miR-197-3p, and among these, hsa-miR-6879-5p and hsa-miR-1260a, which had particularly high expression, were selected as markers (Fig. 22).

[0167] Through this, it was confirmed that the biomarkers for early diagnosis of arteriosclerosis were SERPINA11, F2, AHSG, hsa-miR-143-5p, has-miR-1260a, and has-miR-6879-5p (Fig. 23).

Claims

1. A biomarker composition for diagnosing arteriosclerosis, comprising at least one selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a, and miR-6879-5p.

2. A biomarker composition for diagnosing arteriosclerosis, wherein the arteriosclerosis occurs in the aorta, coronary artery, basilar artery, renal artery, or peripheral artery.

3. A biomarker composition for diagnosing arteriosclerosis, comprising at least one selected from the group consisting of SERPINA11, F2, AHSG, miR-143-5p, miR-1260a, and miR-6879-5p, according to claim 1.

4. In the third paragraph, the biomarker composition for diagnosing arteriosclerosis is a biomarker composition for diagnosing arteriosclerosis for early diagnosis of arteriosclerosis.

5. In the third paragraph, the biomarker composition for diagnosing arteriosclerosis is a biomarker composition for diagnosing arteriosclerosis, the expression of which changes in blood or plasma-derived exosomes when arteriosclerosis develops.

6. In the third paragraph, a biomarker composition for diagnosing arteriosclerosis, wherein the expression of SERPINA11, F2, miR-6879-5p and miR-1260a increases with the onset of arteriosclerosis.

7. A biomarker composition for diagnosing arteriosclerosis, wherein the expression of AHSG and miR-143-5p in the third paragraph is reduced due to the onset of arteriosclerosis.

8. A composition for diagnosing arteriosclerosis, comprising a preparation for measuring the expression level of one or more biomarkers selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a, and miR-6879-5p.

9. A composition for diagnosing arteriosclerosis, wherein the agent for measuring the expression level of the protein biomarker in claim 8 is a peptide, antibody or fragment having immunological activity thereof that specifically binds to the full-length protein or a fragment thereof, an aptamer, an avidity multimer or peptidomimetics.

10. A composition for diagnosing arteriosclerosis, wherein the measurement is performed by one or more methods selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

11. In the 8th paragraph, the agent for measuring the expression level of the microRNA biomarker is a composition for diagnosing arteriosclerosis, which is a PNA (Peptide nucleic acid), aptamer, primer pair, probe, or primer pair and probe that specifically recognizes a nucleic acid sequence of microRNA, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and the complementary sequence.

12. A composition for diagnosing arteriosclerosis, wherein the measurement is performed by one or more methods selected from the group consisting of polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, RNase protection assay (RPA), next generation sequencing (NGS), in situ hybridization, nucleic acid microarray, northern blot, DNA chip, multiplex PCR, or ddPCR.

13. A kit for diagnosing arteriosclerosis comprising the composition of claim 1 or claim 8.

14. A kit for diagnosing arteriosclerosis according to claim 13, wherein the kit is an RT-PCR (Reverse transcription polymerase chain reaction) kit, a DNA chip kit, an ELISA (Enzyme-linked immunosorbent assay) kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

15. A method for providing information for the diagnosis of arteriosclerosis, comprising the step of measuring the expression level of one or more genes selected from the group consisting of RANBP1, MAN1A1, TKFC, UROC1, SERPINA11, F2, AHSG, miR-143-5p, miR-1260a, and miR-6879-5p in a biological sample isolated from an individual.

16. A method for providing information on the diagnosis of arteriosclerosis, wherein the biological sample is an exosome derived from blood or plasma, according to claim 15.

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