KR20140074559A

KR20140074559A - A Kit for Diagnosing the Cardiovascular Disease

Info

Publication number: KR20140074559A
Application number: KR1020120142642A
Authority: KR
Inventors: 양정희; 정광회
Original assignee: (주)차바이오메드
Priority date: 2012-12-10
Filing date: 2012-12-10
Publication date: 2014-06-18

Abstract

The present invention relates to a method for screening a substance for treating or preventing cardiovascular diseases, a diagnostic kit for the cardiovascular diseases, and a pharmaceutical composition for treating the cardiovascular diseases. The screening method of the present invention is able to be useful for screening a substance for down-regulating carboxypeptidase N1 (CPN1), a complement factor B, a fibrinogen beta chain, gelsolin, and complement component 4 binding protein which are overexpressed in cardiovascular disease patients′s plasma. Also, according to the present invention, since CPN1 protein expression is significantly increased in the patient group with ST segment elevation myocardial infarction, fibrinolysis in blood vessels is stimulated by suppressing CPN1 expression using the method and the presence or absence of the ST segment elevation myocardial infarction is able to be checked quickly and easily by measuring expression level of the CPN1.

Description

A kit for diagnosing the cardiovascular disease < RTI ID = 0.0 >

The present invention relates to a cardiovascular disease diagnostic kit.

Coronary artery disease (CAD) is a major cause of cardiovascular disease and death. In the stenosis angiogram analysis, physical stress tests, increased risk factors (eg, increased levels of low density lipoprotein (LDL) / cholesterol, high blood pressure, diabetes, and obesity) were found to be a strong predictor of CAD. However, there is a need for effective methods to manage these high-cost and survival-threatening diseases due to the ever-increasing number of patients. From this point of view, identification of early diagnosis biomarkers of CAD and development of immediate treatment methods for high-risk patients are necessary.

Atherosclerosis has an unstable plaque with a large lipid core and a thin fibrous cap and is formed by regulatory abnormalities of endothelial cells, platelets, smooth muscle cells and leukocyte infiltration (1). The cytokines, growth factors, and matrix metalloproteinases (MMPs) produced by these cells affect the stabilization and degradation of the fibrous cap extracellular matrix (2,3). Recent studies have also shown that plaque rupture of atherosclerosis is inevitably associated with angiographically stenotic sites, and the composition of atherosclerotic plaques plays a more important role in plaque rupture and thrombosis (4). (CRP), fibrinogen, Factor VIIc, Factor VIIIc, plasminogen activator inhibitor-1 (PAI-1) in the study of cardiovascular biomarkers for the early diagnosis of pathologic coronary artery disease in the blood ) Levels, the presence of anti-phospholipid antibodies, and platelets and reactivity (5). Several studies have shown that CRP, an important inflammatory marker, is overexpressed in sera from patients with aberrant atherosclerotic plaques (6), and significantly reduced CRP levels and improved symptoms when treated with simvastatin (7, 8). These results suggest that CRP can be a reliable biomarker and treatment target in CAD patients. Conversely, in some other studies, CRP levels and severity of CAD were found to be less or less significant (9-11).

Proteome-based studies to identify biomarkers have the advantage of post-translational protein processing and mRNA expression associated with protein synthesis. In addition, protein expression pattern analysis that reflects physiological changes is an excellent method for finding biomarkers for diagnosis, prediction, and treatment. Proteomic analysis allows the protein to be separated by molecular weight or isoelectric point using 2-D PAGE, liquid chromatography or mass spectrometry, thereby combining the MALDI-TOF mass spectrometry results and database to identify proteins (12,13).

In order to identify the biomarker of the cardiovascular disease or the therapeutic target, proteomic analysis was performed in order to measure the expression of protein in plasma of CAD patient and normal control group.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to elucidate biomarkers and therapeutic targets for early diagnosis of cardiovascular diseases. As a result, the plasma levels of CPN1 (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, Gelsolin and complement component 4 binding protein) was overexpressed. In particular, the expression of CPN1 (carboxypeptidase N1) was significantly increased. The present inventors have completed the present invention by developing a screening method for a material for treating or preventing a cardiovascular disease, a cardiovascular disease diagnostic kit and a composition for treating cardiovascular disease by utilizing the high expression of the proteins in cardiovascular disease patients as compared with a normal control group .

It is therefore an object of the present invention to provide a method for screening a substance for treating or preventing a cardiovascular disease.

Another object of the present invention is to provide a kit for diagnosing cardiovascular diseases.

It is another object of the present invention to provide a pharmaceutical composition for the treatment of cardiovascular diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a substance for treating or preventing a cardiovascular disease comprising the steps of:

(a) a gene or protein of CPN1 (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, gelsolin or complement component 4 binding protein Contacting the sample to be analyzed with a cell comprising the sample; And

(b) measuring the expression level of the genes, the amount of the protein or the activity of the protein, the expression level of the genes, the amount of the protein or the activity of the protein is down-regulated, The sample is judged to be a substance for the treatment or prevention of cardiovascular disease.

The present inventors have made intensive researches to identify biomarkers and therapeutic targets for the early diagnosis of cardiovascular diseases. As a result, they have found that the plasma of CPN1 (carboxypeptidase N1), complement factor B, fibrinogen beta chain , beta chain, Gelsolin, and complement component 4 binding protein were overexpressed. In particular, CPN1 (carboxypeptidase N1) expression was significantly increased.

There are two types of carboxy peptidases: carboxypeptidase N (CPN) and activated thrombin-activable fibrinolysis inhibitor (TAFIa), which play an important role in plasma. CPN and TAFIa share important substrate specificity including endothelial cell-surface plasminogen / tissue-type plasminogen activator (t-PA) receptors. CPN circulates in the body as a carboxypeptidase N / E family member at a concentration of 100 nM or less and consists of two enzyme active subunits (CPN1) and two regulatory subunits (CPN2). Recent studies have shown that plasmin cleavage increases the catalytic activity of CPN, especially CPN and plasmin-cleaved CPN (CPNc) have anti-fibrinolytic properties.

The present inventors identified nine proteins overexpressed in comparison to the normal control in patients with cardiovascular disease, that is, coronary artery disease. Among them, CPN1 (carboxypeptidase N1), complement factor B, fibrinogen beta chain (fibrinogen, beta chain), gelsolin and complement component 4 binding protein were significantly different from the normal control group. Particularly, it has been found that CPN1 (carboxypeptidase N1) among the five proteins is significantly increased in patients with ST segment elevation myocardial infarction. Considering that overexpression of CPN1 may be involved in fibrinolysis in cardiovascular diseases, A method for screening a preventive substance, a cardiovascular disease diagnostic kit, and a pharmaceutical composition for treating cardiovascular diseases.

The method of screening for a therapeutic or prophylactic agent for cardiovascular diseases according to the present invention is applicable to a variety of known cardiovascular diseases such as myocardial infarction (e.g., ST segment elevation myocardial infarction, ST segment non-elevated myocardial infarction), heart failure (e.g., congestive heart failure) But are not limited to, stroke, arrhythmia (e.g., atrial fibrillation), atherosclerosis, hypertension, aneurysm, embolism, stroke, thrombosis or angina pectoris (e.g., stable angina pectoris, unstable angina pectoris and dysenteric angina pectoris).

According to the method of the present invention, first, a complementary factor 4 binding protein (CPN1), a complement factor B, a fibrinogen, a beta chain, a gelsolin or a complement component 4 binding protein ) Is contacted with a sample to be analyzed. The term "sample" used in referring to the screening method of the present invention includes CPN1 (carboxypeptidase N1), cofactor B ( complement factor 4 binding protein, complement factor B), fibrinogen, beta chain, gelsolin or complement component 4 binding protein, &Lt; / RTI > Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Next, the expression level of CPN1, cofactor B, fibrinogen beta chain, gelsolin or ancillary component 4 binding protein is measured in the sample-treated cells. Various methods known in the art can be used for the measurement of the expression level, for example, a polymerase chain reaction, Luciferase control, ELISA (enzyme immunoassay) and 2D PGAE (2-dimensional polyacrylamide gel electrophoresis) , But is not limited thereto. As a result of measurement of the expression level, the sample can be judged to be a substance for the prevention or treatment of cardiovascular disease when the CPN1, cofactor B, fibrinogen beta chain, gelsolin or auxiliary factor 4 binding protein is inhibited by high yield.

The present invention is based on the correlation between the degree of expression of CPN1, cofactor B, fibrinogen beta chain, gelsolin or ancillary factor 4 binding protein and cardiovascular disease. That is, the expression of genes or proteins of CPN1, cofactor B, fibrinogen beta chain, gelsolin or ancillary factor 4 binding protein appears to be highly expressed in plasma of patients with cardiovascular disease. The correlation of these cardiovascular disease diagnostic molecular markers is very unique compared to conventional cardiovascular disease molecular markers. The term " high expression " as used herein to refer to a gene or protein of CPN1, cofactor B, fibrinogen beta chain, gelsolin or ancillary component 4 binding protein refers to the presence of the gene or protein in the sample And the degree of expression of the protein is higher than that of a normal sample (for example, a cell).

According to a specific embodiment of the present invention, the expression of CPN1, cofactor B, fibrinogen beta chain, gelsolin or auxiliary factor 4 binding protein is analyzed using 2-D gel PAGE or ELISA to determine the presence or absence of cardiovascular disease .

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a carboxypeptidase N1, a complement factor B, a fibrinogen, a beta chain, a gelsolin or a complement component 4 binding protein, or a primer or a probe that specifically binds to a nucleic acid molecule encoding the protein.

The probes or primers used in the diagnostic kit of the present invention may be selected from the group consisting of CPN1, cofactor B (or cofactor B preproprotein), fibrinogen beta chain (or fibrinogen beta chain prepoprotein), gelsolin (or gelsol isoform) Has a sequence complementary to the gene sequence of an accessory factor 4 binding protein (e. G., Cofactor 4 binding protein alpha). The term " complementary ", as used herein, refers to a combination of CPN1, cofactor B (or cofactor B preproprotein), fibrinogen beta chain (or fibrinogen beta chain pre- (E. G., Gelsolin isoform) or a complementary factor 4 binding protein (e. G., Cofactor 4 binding protein alpha). Thus, the term " complementary " has a different meaning from the term perfectly complementary, and the primer or probe of the present invention can be used in combination with CPN1, cofactor B (or cofactor B preproprotein), fibrinogen beta chain (or fibrinogen Beta-chain pre-protein), gelsolin (or gelsolin isoform) or an auxiliary factor 4 binding protein (e.g., cofactor 4 binding protein alpha) May have a mismatch base sequence.

The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin , Haptens with specific binding partners such as biotin, digoxigenin and chelating groups. Markers can be generated using a variety of methods routinely practiced in the art such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

As used herein, the term " substrate "refers to a specific molecule that binds to an enzyme protein, and the enzyme exhibits a specific activity when bound to a substrate having specific affinity. Using such substrate specificity, CPN1 (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, gelsolin or complement component 4 binding protein, Or an antibody can be prepared.

The kit of the present invention may further include other components in addition to the above components. For example, if the kit of the invention applied to a PCR amplification process, the kit of the present invention optionally, reagents required for PCR amplification, for example, buffers, DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus A thermostable DNA polymerase obtained from literalis or Pyrococcus furiosus (Pfu)), may include a DNA polymerase and dNTPs joinja. In addition, when the kit of the present invention is applied to an ELISA assay, the kit of the present invention may optionally comprise reagents required for ELISA analysis, such as CPNl (carboxypeptidase N1), complement factor B, fibrinogen beta chain fibrinogen, beta chain, Gelsolin, a primary antibody specific for the complement component 4 binding protein, a secondary antibody specifically binding to the primary antibody, and a secondary antibody capable of binding to the secondary antibody A labelable substance, and the like. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

The cardiovascular disease diagnostic kit of the present invention may be a PCR amplification kit, a microarray or an ELISA kit, and the diagnostic kit may be applied to biological samples such as whole blood, serum or plasma, and the like.

(A) an inhibitor of CPN1 (carboxypeptidase N1) gene or an inhibitor of CPN1 (carboxypeptidase N1) protein as an active ingredient; And (b) a pharmaceutically acceptable carrier.

The pharmaceutical composition for the treatment of cardiovascular diseases according to the present invention is characterized in that, like the above-mentioned cardiovascular disease diagnosis kit of the present invention, CPN1 (carboxypeptidase N1), complement factor B, fibrinogen beta chain (Fibrinogen, Beta Chain), Gelsolin or Complement Component 4 Binding Protein, the common content of which is to avoid the excessive complexity of the present specification, The description is omitted.

The pharmaceutical composition for the treatment of cardiovascular diseases of the present invention can be used for the treatment of various cardiovascular diseases such as myocardial infarction (for example, ST segment elevation myocardial infarction, ST segment non-elevated myocardial infarction), heart failure (for example, congestive heart failure) (E.g., atrial fibrillation), atherosclerosis, hypertension, aneurysm, embolism, stroke, thrombosis or angina pectoris (e.g., stable angina pectoris, unstable angina pectoris and dysenteric angina pectoris). When the composition of the present invention is administered to a patient, the expression or activity of CPN1 is inhibited to increase fibrinolysis in the cardiovascular system. Accordingly, the pharmaceutical composition of the present invention can be applied not only to cardiovascular diseases, but also to all pharmaceutical compositions used for thrombolytic use of blood vessels.

Inhibitors of CPN1 (carboxypeptidase N1) gene or CPN1 (carboxypeptidase N1) protein contained as an active ingredient in the composition of the present invention are collectively referred to as substances capable of inhibiting expression of CPN1 at DNA, RNA or protein level. In other words, the term " inhibiting expression " is used herein to control the expression of CPN1 protein in the course of mRNA processing, or to suppress the activity of CPN1 protein by controlling CPN1 gene transcription and CPN1 protein expression, Means that the expression / activity of CPN1 is regulated at the level of gene and protein to control the effect of CPN1 inhibiting fibrinolysis. When the expression of CPN1 is suppressed, fibrinolysis in the cardiovascular system can be increased to treat or alleviate symptoms of cardiovascular disease.

The composition of the present invention may contain, as an active ingredient, an inhibitor of CPN1 (carboxypeptidase N1) gene or an inhibitor of CPN1 (carboxypeptidase N1) protein, (a) a complement factor B, a fibrinogen, Inhibitors of the expression of Gelsolin and complement component 4 binding protein genes; And (b) a protein inhibitor of cofactor B, fibrinogen beta chain, gelsolin and cofactor 4 binding protein.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, percutaneous administration, mucosal administration, or topical administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Preferably, the dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for screening a substance for treating or preventing a cardiovascular disease, a cardiovascular disease diagnostic kit, and a pharmaceutical composition for treating cardiovascular diseases.

(b) The method of the present invention is useful for the treatment of cardiovascular disease in a patient suffering from cardiovascular disease, comprising administering to a patient in need thereof a therapeutically effective amount of at least one selected from the group consisting of CPNl (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, Gelsolin, Can be useful in screening for substances that can down-regulate the complement component 4 binding protein.

(c) According to the present invention, protein expression of CPN1 (carboxypeptidase N1) is markedly increased in patients with ST segment elevation myocardial infarction, which can be used to inhibit expression of CPN1 to promote fibrinolysis in blood vessels , The degree of expression of CPN1 can be measured to quickly and easily detect the onset of ST segment elevation myocardial infarction.

Figure 1 shows a 2-D PAGE gel using plasma proteins from patients with coronary artery disease (CAD) and normal control. The gel was stained with Comassi Blue G-250. b, c, d, e, g, l, m; Stable angina, f, i, j, k; Unstable angina, h; ST segment elevation myocardial infarction, a; Normal control.
FIG. 2 shows the results of analyzing the intensity of 9 protein spots in the plasma of CAD patient and normal control after 2-D PAGE. FIG. (A) of the plasma protein spot in the patient group and the normal control group, and the intensity (B) of the plasma protein spot in each patient and each control volunteer. The protein spots showed a intensity of > 1.7. b, c, d, e, g, l, m; Stable angina, f, i, j, k; Unstable angina, h; ST segment elevation myocardial infarction, a; Normal control.
FIG. 3 shows the results of analysis of CPN1 expression in Plasma of CAD patient by ELISA. The CPN1 expression level was 8.7 ± 2.5 ng / ㎖ (n = 29) in the normal control group, 6.7 ± 2.1 ng / ㎖ in the SA patient group and 9.8 ± 2.3 ng / And 50.5 ± 25.3 ng / ㎖ (n = 14) in patients with STEMI (p = 0.001). Data were expressed as means ± standard error, and ELISA samples were analyzed twice.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

Plasma protein sample

Blood samples were collected from 12 patients (7 SA; 4 UA; 1 STEMI) and 9 healthy volunteers (6 males and 3 females) with coronary artery disease at Yonsei University Gangnam Severance Hospital (Seoul, Korea) SA (stable angina), UA (unstable angina), STEMI (ST segment elevation myocardial infarction, ST segment elevation myocardial infarction). The median age of 12 patients with coronary artery disease is 59 years (range, 43-77 years, 9 males and 3 females). Patients and healthy volunteers participated in the study and were approved by the Human Biomedical Research Program Committee at Yonsei University College of Medicine. 10 ml of peripheral blood was collected in a K3EDTA (Ravencart) tube. The tubes were centrifuged at 3,000 rpm for 15 minutes and the supernatant was pooled and transferred to a 15 ml conical tube. To remove unwanted blood proteins, a plasma sample was applied to the MARC column and the eluted protein fraction was precipitated with acetone and used for further analysis. Plasma 0.5 ml was collected from 9 healthy volunteers and pooled and used as a control.

Protein quantification

Plasma proteins were quantified using the Bradford protein assay (Bio-Rad, USA).

2-D PAGE (2-dimensional polyacrylamide gel electrophoresis)

IPG pH 47 non linear DryStrips 24 cm (Genomain, South Korea) were mixed with 7 M urea, 2 M thiourea, 1% w / v DTT, 1% pharmylate and 2% v) rehydrated in a swelling buffer consisting of CHAPS. The plasma protein 80 was transferred to a rehydrated IPG strip and was subjected to isoelectric focusing (IEF) at 20 ° C. The electrophoresis was performed using a Multiphor II device and an EPS 3500 XL power supply GE Healthcare Bio-Science, Sweden) was used and experiments were conducted according to the manufacturer's instructions. After the sample was loaded at isoelectric point electrophoresis, the voltage was continuously increased from 150 V to 3,500 V over 3 hours, and then maintained at 3,500 V. Focusing was completed after 96 kV / h. The strips were placed in an equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% SDS, and 30% glycerol) for 10 min before running the second dimension, Was treated in turn with 2.5% iodoacetamide. The equilibrated strips were inserted into a 1016% gradient SDS-PAGE gel (20 X 24 cm). SDS-PAGE was performed according to the manufacturer's instructions using a Hoefer DALT 2D system (Amersham Biosciences). The 2-D PAGE gel was run at 1,700 Vh at 0 ° C. Thereafter, the gel was fixed for 1 hour in 40% (v / v) methanol containing 5% (v / v) phosphoric acid, and stained with colloidal Coma Blue G-250 solution for 5 hours , Korea) and decolorized in 1% (v / v) acetic acid for 4 hours.

2-D PAGE gel image analysis

Protein spot detection and 2-D pattern matching were performed according to the manufacturer's protocol using PDQuest version 7.0 software (Bio-Rad) (13). Each protein spot was normalized to the intensity of all available spots. All gel spots were normalized by comparing plasma protein spot concentrations between CAD (coronary artery disease patients) and controls. Quantified candidate protein spots were compared by histogram.

In-Gel Digestion and Peptide Extraction with Trypsin

The process of in-gel protein spot degradation was carried out by a known method (13). Approximately, a protein spot stained with coma blue was cut out and scraped. The gel pieces were washed with 25 mM ammonium bicarbonate buffer (pH 7.8, 50% (v / v) acetonitrile, ACN) for 1 hour at room temperature. The gel pieces were dehydrated in SpeedVac for 10 minutes and then rehydrated in 10 μl (20 ng / μl) of sequencing grade trypsin solution (Promega). After treatment for 24 hours at 37 ° C in 25 mM ammonium bicarbonate buffer (pH 7.8), the trypsin-digested peptides were sonicated for 40 minutes in 0.5% TFA (50% (v / v) ACN). The extracted solution was reduced to 1 [mu] l in a vacuum centrifuge. Before mass analysis, the peptide solution was desalted using a reversed-phase column.

The peptide solution was applied to the column after filling with Poros 20 R2 resin (Pearl Spective Biosystems, USA) with GE Loader tip (Eppendorf, Germany) and equilibrium with 10 μl of 5% (v / v) formic acid. It was then washed with 10 [mu] l of 5% (v / v) formic acid. The bound peptides were eluted with 1 [mu] l of alpha -cyano-4-hydroxycinnamic acid (5 mg / ml, 50% (v / v) ACN / 5% (v / v) formic acid) X 2; Applied Biosystems, USA).

MALDI - TOF Using a mass spectrometer Peptides Analysis and protein identification

Mass determinations of trypsin-degrading peptides were performed in the above-described reflectron positive ion mode mode using a Voyager-DE STR mass spectrometer (Pearl Spective Biosystems). Close external calibrations were performed with a calibration mixture for each of the four samples and the calibration mixture was applied to the adrenocorticotropic fragment 1839 (moto isotopic mass, 2465.1989), neurotensin (moto isotopic mass, 1672.9175) and angiotensin I (moto isotopic mass, 1296.6853) were used as standard calibrators. Mass spectrum of 9003500 Da mass range was obtained. Proteins were identified using peptide-mass fingerprinting analysis on the Swiss-Prot and NCBI databases. The analysis program Profound (http://129.85.19.192/profound_bin/WebPro Found.exe, Rockefeller University, version 4.10.5), MASCOT (http://www.matrixscience.com) or MS-Fit (http://prospector.ucsf.edu, University of California San Francisco, Version 4.0.5). Subsequently, mass spectrometry parameters were constructed: peptide mass tolerance 50 ppm; Mass range 0-100 kDa; Allow differentiation, 2; Methionine oxidation, and cysteine thropionamide. Only the portion where at least four peptide masses defined by each program were matched was considered initially.

ELISA Validation analysis

Overexpressed proteins in plasma samples of patients were analyzed by enzyme immunoassay (ELISA) using the corresponding antibodies. All samples were analyzed twice. ELISA kits for transferrin, prealbumin and hemopexin were purchased from Assaypro (USA). ELISA kits for apolipoprotein H (? 2-glycoprotein 1,? 2-GP1) and CPN1 were purchased from US Biological (USA) and Uscn Life Science Inc. (China).

Statistical analysis

Statistical analysis was performed using PASW version 18.0. Statistical comparisons between groups were performed using one-way analysis and the Tukey's test, which was used for post hoc analysis. Data were expressed as mean ± standard error (p <0.05).

Experiment result

Patients with coronary artery disease plasma Proteome profile

2-D PAGE was performed using 80 μg of each plasma protein in 12 patients (7 SA, 4 UA, and 1 STEMI) and 9 healthy volunteers (normal control group). In order to separate the plasma proteins according to molecular weight and isoelectric point, 2-D PAGE was performed using non-linear IPG of pH 47. The gel was then stained with Coma Brilliant Blue G-250. Figure 1 shows 2-D PAGE gel images from SA patients (b, c, d, e, l and m), UA patients (f, i, j and k) and STEMI patients (h), respectively. In order to determine the volumetric intensity of differentially expressed proteins in patient and normal groups, 32 protein spots on 2-D PAGE gels with markedly different expression were found. Of these spots, IN2 (# 800), IN3 (# 905), IN7 (# 1106), IN11 (# 1194), IN13 (# 1737), IN14 # 1215) and IN19 (# 1248) were 1.7 times more overexpressed in the plasma of CAD patients than in the normal control (FIGS. 2a and 2b).

MALDI - TOF Mass analysis

The nine protein spots identified in FIG. 2 were cleaved to perform in-gel trypsin digestion, followed by MALDI-TOF analysis. Peptide mass fingerprinting maps obtained through MALDI-TOF mass spectrometry were used for protein identification. Nine proteins were found to be more potent than the others, including cofactor B preproproteins (IN2, # 800), transferrin (IN3, # 905), apolipoprotein H (IN7, # 1106), fibrinogen beta chain pre- (IN16, # 926), hemopexin (IN18, # 1215) and carboxypeptidase N1 precursor (IN19, # 927), albumin (IN13, # 1737), gelsolin isoform a 1248) (reliability> 95%, anda z -score> 1.76). Information on the identified proteins (protein name, speech number, molecular weight, pI value and increased intensity ratio are shown in Table 1).

Identification of over-expressed proteins in plasmas from CAD patients Spot # protein Session number Top score (Est'd Z) Number of matched peptides Sequence Coverage (%) Mr (KDa) /
pI burglar IN2 ^†
(# 800) Cofactor B preproprotein NP_001701.1
2.38 18/53 26 99 / 6.5
+2.6
IN3
(# 905) Transferrin NP_001754.1
2.39 33/59 48 75 / 6.8 +4.4 IN7 ^†
(# 1106) Chain A, crystal structure of human beta-2-glycoprotein-I (apolipoprotein H) 1C1Z | A 2.36 10/24 48 57 / 6.3 +2.1 IN11
(# 1194) Fibrinogen beta chain pre-protein NP_005132.1 2.41 25/62 44 53.5 / 6.4 +1.8 IN13
(# 1737) Prealbumin NP_000362.1
1.76 7/25 64 16 / 5.3 +2.1 IN14
(# 1992) Gelsolin isoform a NP_000168.1 2.42 19/77 30 85 / 4.7 +2.0 IN16
(# 926) Cofactor 4 binding protein alpha NP_000706.1
2.36 13/50 33 70 / 6.2 +1.8 IN18
(# 1215) Hemopexin NP_000604.1 2.39 11/31 29 52 / 6.6 +1.7 IN19
(# 1248) Carboxy peptidase N, polypeptide 1 50 kD precursor NP_001299.1 2.15 10/45 25 51 / 6.4 +2.1

* (+); Represents the increased percentage of protein spot intensity in the CAD patient plasma as compared to the normal control. ^{† Represents} a protein spot that is likely to deform.

ELISA Validation Research

In order to validate proteomic analysis, ELISA was performed using plasma samples of the corresponding antibodies and CAD patients. Plasma samples were collected from 29 normal controls, 30 SA, 31 UA and 14 STEMI patients. Patient information is shown in Table 2. Overexpression of transferrin, prealbumin, hemopexin, or apolipoprotein H was not associated with CAD severity. However, the overexpression of CPN1 in the STEMI patient plasma was dramatically higher than in the normal control, SA or UA. (N = 30) in SA patients and 9.8 ± 2.3 ng / ㎖ (n = 29) in UA patients in the normal control group, while the expression level of CPN1 was 8.4 ± 2.5 ng / 31) and 50.5 ± 25.3 ng / ㎖ (n = 14) in STEMI patients (p = 0.001) (Fig. 3).

Post hoc analysis by the Tukey's test showed that the expression of CPN1 was significantly increased in STEMI patients compared to the other groups (p <0.05). Therefore, the present inventors intend to present CPN1 as a diagnostic biomarker of STEMI through validation studies using a large number of sample sets.

Patient information of samples used in ELISA analysis of Carboxypeptidase N1 (CPN1) - Control (n = 29) SA (n = 30) UA (n = 31) STEMI (n = 14) age 57.6 ± 11.2 63.9 ± 9.3 64.8 ± 10.3 56.8 ± 11.4 gender 15/14 16/14 19/12 13/1 Risk factor High blood pressure 15 25 19 5 smoking 11 13 14 11 diabetes 5 12 6 4 Dyslipidemia 4 5 6 2 Stent vasodilation One One 10 0 Medication ACE inhibitor 10 24 24 12 Beta inhibitor 9 18 18 10 Calcium antagonist 16 8 11 0 nitrate 9 10 23 12 Statin 14 26 29 13 aspirin 12 27 27 14 Heparin 0 3 2 10 Disgren 0 0 11 0

* AEC inhibitors; Angiotensin-blocking enzyme inhibitor

Argument

Although a rupture of atherosclerotic plaque causes a fatal acute arterial syndrome, in many cases it has not shown any obvious clinical symptoms (14). In the present invention, it was found that fibrinogen beta chain pre-protein, cofactor B pre-protease, C4BPA, apolipoprotein H, gelsolin isoform a and CPN1 were expressed 1.7 times higher than plasma of CAD patient group compared with normal control group . In a CAD biomarker study by proteomic analysis, Mateos-Caceres et al. Found that fibrinogen gamma-chain, as well as ATT, apolipoprotein A1, immunoglobulin D heavy chain and albumin, were differentially expressed in plasma of acute CAD patients 15). In urinary samples of CAD patients, it was also found that fibrinogen gamma-chain expression increases with increasing short sides of collagen alpha I and III (16). Similarly, 'Badinon et al.' And 'Danesh et al.' Have shown that high fibrinogen concentrations in plasma are associated with an increased risk of cardiovascular disease and are associated with precipitated and increased fibrin thrombosis at lesion sites (4, 17). This is also evidenced by the overexpression of fibrinogen in the CAD patients of the present invention, and this overexpression is associated with CAD.

The degree of inflammation by immunoregulatory cells in CAD progression is described in Hansson et al. And Libby et al. (18, 19). Similarly, in the present invention, cofactors such as cofactor B and C4BPA were overexpressed in CAD patients. In a recent study by Speidl, MMP-1 and MMP-9 levels were significantly elevated when monocytes-macrophages were stimulated with C5a and were found to be present on human coronary artery plaques (20). Furthermore, the expression of C5a receptor was higher in the normal diet than in the control group in ApoE - / - mice. As a result, when treated with C5a receptor antagonist, the lesion site was reduced and lipid content was reduced in ApoE - / - mice (21). These results suggest that an increase in cofactor B levels plays an important role in CAD progression through C5a. In addition, the increase in data related to these data supports the important role of apolipoprotein-H in CAD. In accelerated atherosclerosis, thrombosis in anti-phospholipid syndrome (APS) where complexes of anti-phospholipid antibodies, anti-cardiolipin antibodies and anti-β-2GP1 (apolipoprotein-H) Proliferation was predominant (22). In addition, recent clinical studies have shown that acute MI and heart attack account for 30% of APS patient deaths (23), which implies a broad functional range of apolipoprotein-H in atherosclerosis and thrombosis. However, some studies have shown controversy regarding the association of apolipoprotein-H expression and CAD lesions. Indeed, the protective function of apolipoprotein-H is required in the progression of atherosclerosis and the greatest reduction in apolipoprotein-H levels in patients exhibiting the highest CRP levels, which is due to the interaction between platelets and endothelial cells (24, 25). To confirm the association of apolipoprotein-H and CAD, we performed ELISA analysis using plasma samples from 51 healthy individuals, 37 SA patients, 18 UA patients and 7 STEMI patients. Apolipoprotein-H levels were 306 ± 29.9 ㎍ / ㎖ in normal control group, 588 ± 247.0 ㎍ / ㎖ in SA group, 346 ± 20.9 ㎍ / ㎖ in UA group and 370 ± 46.3 ㎍ / ㎖ in STEMI group (p = 0.5). However, there were no statistically significant differences in apolipoprotein-H expression in the CAD patient group and the normal group.

The present inventors firstly confirmed that CPN1 is overexpressed in the plasma of CAD patients through proteome analysis. Similarly, in ELISA validation studies CPNl levels were significantly increased in plasma from STEMI patients (Figure 2). To analyze the importance of CPN1 overexpression in patients with STEMI, we focused on the role of CPN1 in the fibrinolytic pathway, which suggests that thrombosis is dependent on the complex balance between fibrinolysis and fibrinolysis . The formation of occlusive thrombosis is a key feature in STEMI patients, and the degradation of fibrin polymer by plasmin is significantly reduced in CAD and other signs of atherosclerosis (26,27). To date, the carboxypeptidase U or B (thrombin-activated fibrinolysis inhibitor, TAFIa) has been known to play an important role only as a fibrin inhibitor. TAFIa cleaves the C-terminus of the Lys residue on the fibrin, disrupting the binding of plasminogen on the fibrin thrombus, thereby inhibiting plasmin and degrading the fibrin thrombus (28). However, recent research by Walker et al. Suggested that CPN is involved in thrombolysis and is actively involved in the stabilization of the fibrin network, similar to other carboxy peptidase family members (29). Similarly, high CPN1 levels on the plasma in STEMI patients demonstrate stabilization of the fibrin network; Thus, the measurement of CPNl level can be a standard thrombolytic parameter for CAD patients. In addition, a study of CPN1 function in the cardiovascular system of 'Du et al.' Indicates that CPN1 functions as an inflammatory-immunomodulator capable of activating chemerin, a potent chemoattractant. Chemerin binds to chemerin receptors present on the surface of endothelial cells or immune cells and accumulates in the platelets and alleviates platelet activation (30, 31). White agglutination of platelets is prominent in arterial thrombosis, and a closed labeling between CPN1, chemerin, and platelets appears to contribute to CAD progression (32-34).

As a result, the present invention is the first to disclose that CPN1 levels are increased in plasma of STEMI patients, and we present CPN1 as a new diagnostic marker and therapeutic target of STEMI.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

References

1. Naghavi M, Libby P, Falk E, et al. From vulnerable plaques to vulnerable patients: a call for new definitions and risk assessment strategies: Part II. Circulation 2003; 108: 1772-8.

2. Lundberg AM, Hansson GK. Innate immune signals in atherosclerosis. Clin Immunol; 134: 5-24.

3. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (2). N Engl J Med 1992; 326: 310-8.

4. Badimon JJ, Ibanez B, Cimmino G. Genesis and dynamics of atherosclerotic lesions: implications for early detection. Cerebrovasc Dis 2009; 27 Suppl 1: 38-47.

5. Feinbloom D, Bauer KA. Assessment of hemostatic risk factors in predicting arterial thrombotic events. Arterioscler Thromb Vasc Biol 2005; 25: 2043-53.

6. Tanaka A, Shimada K, Sano T, et al. Multiple plaque rupture and C-reactive protein in acute myocardial infarction. J Am Coll Cardiol 2005; 45: 1594-9.

7. Ridker PM, Cannon CP, Morrow D, et al. C-reactive protein levels and outcomes after statin therapy. N Engl J Med 2005; 352: 20-8.

8. Ridker PM, Danielson E, Fonseca FA, et al. Reduction of C-reactive protein and LDL cholesterol and cardiovascular events after initiation of rosuvastatin: a prospective study of the JUPITER trial. Lancet 2009; 373: 1175-82.

9. Reifenberg K, Lehr HA, Baskal D, et al. Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer? Arterioscler Thromb Vasc Biol 2005; 25: 1641-6.

10. Teupser D, Weber O, Rao TN, Sass K, Thiery J, Fehling HJ. No reduction of atherosclerosis in C-reactive protein (CRP) -deficient mice. J Biol Chem; 286: 6272-9.

11. Matsuda S, Yamashita A, Sato Y, et al. Human C-reactive protein enhances thrombus formation after neointimal balloon injury in transgenic rabbits. J Thromb Haemost; 9: 201-8.

12. Zhu H, Bilgin M, Snyder M. Proteomics. Annu Rev Biochem 2003; 72: 783-812.

13. Jeong HC, Kim GI, Cho SH, et al. Proteomic analysis of human small cell lung cancer tissues: up-regulation of coactosin-like protein-1. J Proteome Res; 10: 269-76.

14. Chesebro JH, Badimon L, Fuster V. New approaches to treatment of myocardial infarction. Am J Cardiol 1990; 65: 12C-19C.

15. Mateos-Caceres PJ, Garcia-Mendeza, Lopez Farrea, et al. Proteomic analysis of plasma from patients during an acute coronary syndrome. J Am Coll Cardiol 2004; 44: 1578-83.

16. Delles C, Schiffer E, von Zur Muhlen C, et al. Urinary proteomic diagnosis of coronary artery disease: identification and clinical validation in 623 individuals. J Hypertens; 28: 2316-22.

17. Danesh J, Lewington S, Thompson SG, et al. Plasma fibrinogen levels and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis. JAMA 2005; 294: 1799-809.

18. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352: 1685-95.

19. Libby P, Ridker PM, Hansson GK. Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol 2009; 54: 2129-38.

20. Speidl WS, Kastl SP, Hutter R, et al. The complement component C5a is present in human coronary lesions in vivo and induces expression of MMP-1 and MMP-9 in human macrophages in vitro. FASEB J; 25: 35-44.

21. Manthey HD, Thomas AC, Shiels IA, et al. Complement C5a inhibition reduces atherosclerosis in ApoE - / - mice. FASEB J; 25: 2447-55.

22. Ben-Ami D, Bar-Meir E, Shoenfeld Y. Stenosis in antiphospholipid syndrome: a new finding with clinical implications. Lupus 2006; 15: 466-72.

23. Espinosa G, Cervera R. Morbidity and mortality in the antiphospholipid syndrome. Curr Opin Pulm Med 2009; 15: 413-7.

24. Groot E, de Groot PG, Fijnheer R, Lenting PJ. The presence of active von Willebrand factor under various pathological conditions. Curr Opin Hematol 2007; 14: 284-9.

25. Lin F, Murphy R, White B, et al. Circulating levels of beta2-glycoprotein I in thrombotic disorders and inflammation. Lupus 2006; 15: 87-93.

26. Gebbink MF, Bouma B, Maas C, Bouma BN. Physiological responses to protein aggregates: fibrinolysis, coagulation and inflammation (new roles for old factors). FEBS Lett 2009; 583: 2691-9.

27. Meltzer ME, Doggen CJ, de Groot PG, Rosendaal FR, Lisman T. The impact of the fibrinolytic system on the risk of venous and arterial thrombosis. Semin Thromb Hemost 2009; 35: 468-77.

28. Gils A. Which carboxypeptidase determines the antifibrinolytic potential? J Thromb Haemost 2008; 6: 846-7.

29. Walker JB, Binette TM, Mackova M, Lambkin GR, Mitchell L, Bajzar L. Proteolytic cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity. J Thromb Haemost 2008; 6: 848-55.

30. Du XY, Zabel BA, Myles T, et al. Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable fibrinolysis inhibitor), and platelets. J Biol Chem 2009; 284: 751-8.

31. Goralski KB, McCarthy TC, Hanniman EA, et al. Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. J Biol Chem 2007; 282: 28175-88.

32. Franchini M, Mannucci PM. Venous and arterial thrombosis: different sides of the same coin? Eur J Intern Med 2008; 19: 476-81.

33. Jerjes-Sanchez C. Venous and arterial thrombosis: a continuous spectrum of the same disease Eur Heart J 2005; 26: 3-4.

34. Kumara, Cannon CP. Acute coronary syndromes: Diagnosis and management, part II. Mayo Clin Proc 2009; 84: 1021-36.

Claims

A method of screening for a therapeutic or prophylactic agent for cardiovascular disease comprising the steps of:
(a) a gene or protein of CPN1 (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, gelsolin or complement component 4 binding protein Contacting the sample to be analyzed with a cell comprising the sample; And
(b) measuring the expression level of the genes, the amount of the protein or the activity of the protein, the expression level of the genes, the amount of the protein or the activity of the protein is down-regulated, The sample is judged to be a substance for the treatment or prevention of cardiovascular disease.

The method according to claim 1, wherein the cardiovascular disease is myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, thrombosis or angina.

3. The method of claim 2, wherein the cardiovascular disease is myocardial infarction.

4. The method of claim 3, wherein the myocardial infarction is ST segment elevation myocardial infarction.

The substrate or antibody of CPN1 (carboxypeptidase N1), complement factor B, fibrinogen, beta chain, Gelsolin or complement component 4 binding protein , Or a primer or a probe that specifically binds to a nucleic acid molecule encoding the protein.

The kit for diagnosing cardiovascular diseases according to claim 5, wherein the cardiovascular disease is myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, thrombosis or angina.

The kit for diagnosing cardiovascular diseases according to claim 6, wherein the cardiovascular disease is myocardial infarction.

8. The kit for diagnosing cardiovascular diseases according to claim 7, wherein the myocardial infarction is ST segment elevation myocardial infarction.

10. The composition of claim 9, wherein the composition inhibits expression or activity of CPN1 to increase fibrinolysis in the cardiovascular system.

10. The composition for treating cardiovascular diseases according to claim 9, wherein the cardiovascular disease is myocardial infarction, heart failure, arteriosclerosis, arrhythmia, atherosclerosis, hypertension, aneurysm, embolism, stroke, thrombosis or angina.

12. The composition for treating cardiovascular diseases according to claim 11, wherein the cardiovascular disease is myocardial infarction.

13. The composition for treating cardiovascular diseases according to claim 12, wherein the myocardial infarction is ST segment elevation myocardial infarction.

10. The composition of claim 9, wherein the composition comprises (a) a complement factor B, a fibrinogen, a beta chain, a gelsolin, and a complement component 4 binding protein gene ; And (b) a protein inhibitor of cofactor B, fibrinogen beta chain, gelsolin, and cofactor 4 binding protein.