KR20150009657A - composition for diagnosing coronary artery disease comprising CD31 or fragment thereof - Google Patents

Abstract

The present invention relates to a marker for diagnosing coronary artery disease including CD31 or a fragment thereof. More specifically, when expression level of CD31 or the fragment thereof in a biological specimen is high, entities of the relevant specimen can be diagnosed as an entity which has coronary artery disease or has high possibility of getting coronary artery disease in the future.

Description

A composition for diagnosing coronary artery disease comprising CD31 or a fragment thereof,

The present invention relates to a marker for diagnosing coronary artery disease comprising CD31 or a fragment thereof, and more particularly, to a marker for diagnosing coronary artery disease comprising CD31 or fragment thereof, more specifically, when the expression level of CD31 or a fragment thereof in a biological sample is high, It can be diagnosed as an individual having a high possibility of getting the disease.

Many experimental biomarkers for cardiovascular disease, such as troponin, C-reactive protein (CRP), creatine phosphokinase of muscle band (CPK-MB), brain natriuretic peptide (BNP) and pregnancy- Although proposed, these markers still have limitations. In many cases, troponin has low or negative results in high-risk patients because of its sensitivity to the assay. In addition, CRP and BNP have low diagnostic specificity. Therefore, an early stage specific biomarker that can promote treatment and diagnosis is required.

Circulating endothelial cells (CECs) are known as non-hematopoietic cells and may be referred to as the endothelial marker CD146. Cells originate from damaged blood vessels, are used noninvasively, and are used as important markers for vascular injury, dysfunction, and remodeling. In healthy people, CEC appears to be low. However, CEC appears to be high in a variety of pathologic populations, such as coronary heart disease, immune disease, and infectious disease. According to recent reports, CD146 is expressed on T lymphocytes, but not on hepatocytes or CEC.

Inflammation in coronary artery disease (CAD) provides extensive evidence. Systemic inflammatory responses are known to play a major role in the development of atherosclerosis and myocardial infarction. Immune cells such as monocytes and lymphocytes are involved in this process. These inflammatory cells secrete ROS (Reactive oxygen species) or proteolytic enzymes and act as potential mediators of vascular injury.

The present inventors have studied an effective marker capable of predicting the diagnosis or prognosis of coronary artery disease, and confirmed that the expression level of CD31 is related to the incidence of coronary artery disease, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a composition for the diagnosis of coronary artery disease (CAD) comprising CD31 or a fragment thereof.

Another object of the present invention is to provide a method for detecting a CD31 or a fragment thereof, comprising the steps of (a) immobilizing a biological sample and a control sample to a solid body, (b) treating the solid body with an antibody specifically binding to CD31 or a fragment thereof, (b); and (d) comparing the degree of labeling of the marker of step (c) in the biological sample and the control. And to provide a detection method of the same.

It is another object of the present invention to provide a diagnostic kit for coronary artery disease comprising an antibody or an aptamer that specifically binds to CD31 or a fragment thereof.

In one aspect, the invention relates to a composition for the diagnosis of coronary artery disease (CAD) comprising CD31 or a fragment thereof.

The 'CD31' of the present invention is an abbreviation of 'cluster of differentiation 31', and CD31 is also known as platelet endothelial cell adhesion molecule (PECAM-1). It is one of the human proteins encoded by the PECAM1 gene present on chromosome 17. It is known to perform the role of removing aged neutrophils in the human body. It is involved in intercellular junctions of endothelial cells, and is also expressed in neutrophils, monocytes, natural killer cells, hematopoietic stem cells and certain lymphocyte subgroups. Although the fact that CD31 is an angiogenic marker in healthy individuals has been reported by the present inventors, the characteristics of CD31-expressing cells derived from CAD are not known. The relationship between the degree of CD31 expression and CAD disease is not known at all.

The coronary artery disease of the present invention is a disease caused by an abnormality in the flow of the coronary artery, which is an artery supplying blood to the heart itself, and is usually caused by intraluminal lesions resulting in luminal stenosis and ischemia of the myocardium. Typical diseases include angina pectoris, myocardial infarction, and atherosclerosis. Examples of the coronary artery disease of the present invention include, but are not limited to, angina pectoris or myocardial infarction.

The CD31 marker of the present invention can determine the diagnosis and prognosis of coronary artery disease by confirming the level of CD31 expression in a biological sample. In particular, the present invention relates to a marker capable of diagnosing the possibility of developing coronary artery disease in an individual having a frequency-changing angina, and a diagnostic composition containing the marker.

The present inventors divided into two groups according to the pattern of occurrence of coronary artery disease, one of which was an effort-related angina group, in which a change in clinical pattern in chest pain for 2 months before the experiment (Stable Angina), and the other group has frequency-variable chest pain. Unstable Angina (UA) is a group of patients with newly developed angina, Quot;). The composition of the present invention is a composition comprising a marker capable of diagnosing coronary artery disease in the latter group having frequency-variable chest pain or predicting the possibility of developing coronary artery disease in the future.

(B) treating an antibody that specifically binds to CD31 or a fragment thereof to the immobilization body; (c) immobilizing an antibody that specifically binds to CD31 or a fragment thereof; (c) ) Treating the marker capable of detecting the antigen-antibody binding of step (b); and (d) comparing the level of labeling of the marker of step (c) in the biological sample and the control. Or a method for detecting a fragment thereof.

The biological sample of the present invention is a sample isolated from a subject in which the presence or prognosis of coronary artery disease is to be determined. The biological sample includes tissues, cells, blood, serum, peritoneal fluid, synovial fluid, saliva, urine, It is not. Preferably blood, and may be peripheral blood which is easy to obtain in blood. The control sample can also be a comparative sample, and may be a sample separated from a healthy individual and includes all of the above-mentioned samples.

The antibody that specifically binds to CD31 or a fragment thereof that can be used in the present invention can be easily produced using a method known in the art. To further confirm whether the treated antibody binds to the antigen-antibody, it includes an additional marker treatment step, which includes a secondary antibody conjugate to which a specific marker is bound, a coloring reagent capable of performing a color reaction with the marker, A washing solution and an enzyme reaction stop solution, and the like.

As a representative marker, there is a secondary antibody that binds to the antibody, and usually includes a marker for confirming whether or not the reaction occurs. Examples of such markers include fluorescence such as horseradish peroxidase (HRP), alkaline phosphatase, coloid gold, poly-L-lysine-fluorescein isothiocyanate (RITC), and rhodamine-B-isothiocynate Materials and pigments can be used.

The coloring reagent can be appropriately selected according to the label. For example, TMB (3,3 ', 5,5'-tetramethyl bezidine), BTS [2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid], OPD (o-phenylenediamine), and the like.

In addition, it is preferable that the washing solution includes a phosphate buffer solution, NaCl and Tween-20. After the antigen-antibody binding reaction, the secondary antibody is reacted with the binding agent through the binding reaction, and then an appropriate amount is added to the solid material and washed .

As the reaction stop solution, a sulfuric acid solution may be used.

By performing the antigen-antibody binding reaction as described above to confirm the degree of binding, the expression level of CD31 or fragment thereof in the biological sample can be measured. By comparing the measured expression amount with the amount of the control sample, It is possible to judge whether or not the disease invention is dangerous. If the level of CD31 expression in a biological sample of a subject suspected of coronary artery disease is higher than the level of control CD31 expression, then the individual providing the biological sample may be diagnosed as having a coronary artery disease, or a subject expected to have coronary artery disease As shown in FIG. The present inventors have measured CD31 expression levels in a group with a frequency-variable chest pain (UA) and found CD31 expression to be higher than that in a normal population or an effort-related angina group (SA) .

In another aspect, the present invention relates to a diagnostic kit for coronary artery disease comprising an antibody or an aptamer that specifically binds to CD31 or a fragment thereof.

The above-mentioned antibodies and aptamers can be easily prepared by conventional methods used in the art. The aptamer is a single-stranded nucleic acid molecule having a stable tertiary structure and capable of binding to surface molecules with high affinity and specificity to be.

The diagnostic kit may further include a marker for determining the degree of expression. As mentioned above, the second antibody conjugate conjugated with a commonly used marker substance, a substrate for inducing color development through reaction with the marker substance A reagent, a washing solution, and the like.

As an example of a method of detecting CD31, a separated biological sample is fractionated by SDS-PAGE to CD31 or a fragment thereof, transferred to a fixed body, treated with an antibody specific for CD31 or its fragment to induce binding, The expression level of the fragment can be measured.

As examples of the fixture, a nitrocellulose membrane, a polyvinylidene difluoride membrane, a 96-well plate synthesized with a polyvinyl resin or a polystyrene resin, and a glass slide glass can be used.

In addition, the antigen-antibody binding reaction can be carried out by conventional ELISA (enzyme immunoassay), RIA (radioimmunoassay), sandwich ELISA, Northern blot, Western blot, immunoprecipitation, immunohistochemical staining, , Antigen-antibody aggregation, surface plasmon resonance (SPR), biochip, RNA chip, electrophoresis, PCR, RT-PCR and the like. It is not.

As described above, the present invention can be used as an effective marker for diagnosing or predicting coronary artery disease (CAD) by comparing and comparing the expression level of CD31.

Figure 1 is a fluorescence photograph and graph showing EPC generation, migration, adhesion and anti-apoptotic activity:
[Binding of acetylated low-concentration lipoprotein (red fluorescence) absorption and UEA-1 lectin (green fluorescence) was observed in EPC (A) and EPC colony (B), and C- EPC colonies were generated (n = 5 per group); In chemotaxis analysis (C) and adhesion level analysis (D), C-CD31 cells showed lower mobility and cohesive power than H-CD31 cells (n = 5 per group); Cell apoptosis assays were analyzed using a fluorescent-activated cell sorter (FACS); Both annexin V + and PI + levels were significantly higher in C-CD31 cells than in H-CD31 cells (n = 5 per group).
Figure 2 shows the results of measuring gene expression associated with neovascularization, chemotaxis, anti-apoptotic and inflammatory properties:
[The expression levels of various angiogenic factors (A), chemokines (B), chemokine receptors (C), anti-apoptotic (D) and inflammatory factors in C-CD31 and H-CD31 were measured by RT-PCR (N = 6 per group)].
Figure 3 shows the result of measuring the low therapeutic effect of C-CD31 on the ischemic hind limb:
[(A) The extent of vascular perfusion recovery in the ischemic hind limb was represented by laser Doppler perfusion images;
(B) Quantitative analysis of low blood perfusion in C-CD31 group compared to H-CD31 group and PBS group after 21 days of cell transplantation;
(C) Capillary density in hind limb muscles. The hindlimb muscles were stained with ILB4 (Isolectin B4) to quantitatively analyze the capillary density;
(D) H-CD31 group (n = 8, Bar: C, 50 μm per group) significantly decreased the degree of capillary growth in the C-CD31 group;
(E) Representative images of the ischemic hindlimb region after 4 weeks of transplanted cell lines are shown in the photographs;
(F) The number of cells transplanted from the ischemic legs after 4 weeks of cell injection was measured (DAPI (blue fluorescence) staining of nuclei (n = 5, Bar: E, 100 μm per group).
FIG. 4 is a graph showing the results of analysis of the degree of association between coronary artery and cardiovascular risk disease and CD31 + cell cycle: [(A) Comparing CD31 + cell cycle of SA and UA group versus healthy control group; Correlation of CD31 + cell circulation with coronary artery disease (stenosis> 50%) incidence (B) and individual cardiovascular risk factor (C) in SA or UA patients and healthy control groups.
FIG. 5 is a graph of the number of CD31 + cells estimated for the UA diagnosis through the ROC curve (the area under the curve represents the accuracy of the test and the cut-off value at the CD31 + cell level was 18.5% (95% 21.5)].
Figure 6 shows RT-PCR analysis of high inflammatory characteristics of human peripheral blood-derived CD31 + cells. (A) Expression of major inflammatory genes in peripheral blood-derived and (B) bone marrow-derived CD31 + and CD31- Were analyzed by RT-PCR (n = 4 per group)].

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[Experimental Example]

Experimental Example 1. Selection of experiment group

A total of 78 patients (48 CAD patients and 30 control subjects) were enrolled in this study. CAD, metabolic disease, and inflammatory disease were selected as the control group. SA was defined as effort-related angina, which means that there was no change in clinical pattern in chest pain over the previous 2 months. UA is defined as a group with chest pain with an altered frequency, which refers to a group with a newly developed angina, or a group with mild angina pectoris or resting angina. The degree of disease was calculated as the number of times the coronary artery was affected. The criteria for inclusion in the study were SA and UA patients, ranging from 20 to 80 years of age. Patients with acute myocardial infarction were excluded from this study. The criteria excluded in this study were chronic renal failure (a group with a serum creatinine level greater than 1.4 mmol / L), inflammatory disease, autoimmune disease, anemia (a group with a hemoglobin concentration lower than 8.5 g / dL), and thrombocytopenia L), which has a clinical basis. Details of the clinical features are described in Table 1 below.

All experiments were conducted within the ethical limits allowed by the Institutional Review Board (IRB) of the Dong-A University Medical Center.

[Table 1] Clinical characteristics of patients

Experimental Example 2. CAD Risk Factor

CAD Considered as risk factors were: diabetes (use of insulin or antidiabetic agents), hypertension (repeated hypertension or taking antihypertensive drugs), smoking (more than two per year), and family history Occation). CAD risk factors were measured over 65 years of age, male, smoking, hypertension, diabetes and family history.

Experimental Example 3: CD31 + cell culture and separation

Peripheral blood (PD) was obtained from the patient population and from healthy individuals. Human bone marrow (BM) was purchased from Lonza (Walkersville, MD, USA). The MNCs were separated using known methods. Briefly, each peripheral blood sample was diluted with PBS (phosphate-buffered saline) equivalent volume containing 2% bovine serum albumin (Miltenyi Biotec, Auburn, CA, USA) without calcium and magnesium, and Histopaque-1077 Sigma, St. Louis, Mo., USA) and centrifuged at 1600xg for 20 minutes. The MNCs were collected from the interface and washed with MACS buffer (Miltenyi Biotec). The number of MNCs was then counted and resuspended in the MACS buffer blocking reagent. Human CD31 microbeads (Milteny Biotec) were sequentially added and cultured at 4 占 폚 for 15 minutes. MNCs were washed and resuspended in MACS buffer. CD31 + and CD31- cells were isolated using a magnetic LS column (Miltenyi Biotec) according to the manufacturer's method.

Experimental Example 4. EPC Culture Analysis

EPC (endothelial precursor cell) culture assays were performed using previously known methods. To determine the potential producibility of EPCs, cells were cultured for 4 days at ¹ × 10 ⁶ cells / cm ² on 0.1% fibronectin / gelatin-coated plates in EGM-2 complete medium (Lonza, Walkersvile, MD, USA). The cells were incubated with Dil-acLDL (1: 500, Biomedical Technologies, Staughton, MA, USA) for 1 hour, washed with PBS, fixed with 1% paraformaldehyde, and incubated with FITC-conjugated UEA (Ulex europaeus agglutinin1) -1 < / RTI > lectin (1: 200, Sigma). The number of UEA-1 lectin-binding double-positive cell colonies that have absorbed Dil-acLDL was measured. More than 30 cells were observed as double-positive aggregate EPC colonies.

Experimental Example 5. Statistical Analysis

All data are expressed as mean ± SD unless otherwise noted. And SPSS program package (SPSS version 12.0; SPSS, Chicago, IL, USA). Differences between the two groups were compared using Student's t-testing. Groups were compared using unidirectional ANOVA (analysis of variance). To determine the dispersion heterogeneity, non-parametric Nann-Whitney U test was used. In addition, categorical variables were compared using Pearson's X2 test. Correlations between variables were calculated using Spearman's method. The receiver operating characteristic (ROC) curve interval was used to determine the sensitivity and specificity of CD31-expressing cells to diagnose UA. The optimal cut-off value of CD31-expressing cells was determined. The two-tailed P <0.05 level considered statistical significance for all tests.

Example

Example 1. Characteristics of patient group

A total of 78 individuals were analyzed, and their basic characteristics are as described in Table 1 above. CAD patients were treated with prescriptions such as ACE-inhibitors, aspirin, calcium inhibitors, clopidogrel, diuretics and statins, as well as risk factors such as age, men and diabetes were significantly higher appear.

Example 2. EPC formation, cell migration, adhesion, viability damage of CAD patient-derived CD31 + cells

To determine whether CAD patient-derived CD31 + cells (C-CD31) are damaged during EPC formation, cell migration, adhesion and anti-apoptosis, immunomagnetic separation methods are used CD31 + cells were isolated, and EPC production, chemotaxis, adhesion and apoptosis analysis were performed. EPC analysis showed that C-CD31 was significantly reduced in the EPC and EPC colony passages relative to CD31 + cells (H-CD31) derived from healthy control populations (p = 0.002, P < 0.013, ).

For chemotaxis analysis, VEGF-A was treated with 100 ng / ml and cultured for 24 hours. C-CD31 cell exposure for 24 hours was significantly reduced compared to H-CD31 (P = 0.003) (see FIG. 1C).

In addition, adhesion and apoptosis analysis were performed to investigate cell angiogenesis and anti - apoptotic functions. A large number of cells were observed to attach to extracellular matrix protein collagen I, which was significantly less in the C-CD31 group than in the H-CD31 group (P <0.001) 1D). However, once apoptotic cells were induced, more apoptotic cells were observed in the C-CD31 group than in the H-CD31 group (see FIG. 1E).

Example 3. Inhibition of angiogenesis, co-expression, and anti-apoptotic activity of C-CD31 cells

In order to analyze angiogenesis of C-CD31, expression pattern of various angiogenic factors was measured by real-time RT-PCR. As a result, it was found that (Ang) -1 (angiopoietin, angiopoietin-1), (FGF) -2 (fibroblast growth factor-2), HGF (hepatocyte growth factor) (Epidermal growth factor) were down-regulated to a high level in the C-CD31 group as compared to the H-CD31 group (see FIG. 2A).

To analyze chemokine and inflammatory properties, expression patterns of chemokines and inflammatory factors were analyzed using real-time RT-PCR. It was confirmed that MCP-1 (monocyte chemoattractant-1) and SDF-1a (stromal cell-derived factor-1a) were significantly down-regulated in the C-CD31 group as compared to the H-CD31 group (see FIG. However, the chemokine receptor CXCR2 and inflammatory factor IL-1a were significantly up-regulated in the C-CD31 group (P = 0.001) (see Figures 2C and 2D).

In contrast, a representative anti-apoptotic-related factor AKT-1 was found to be significantly down-regulated in the C-CD31 group (P = 0.009) (see FIG.

Example 4. Low therapeutic effect on ischemic hindlimb

To analyze the therapeutic potential of C-CD31, C-CD31, H-CD31 cells and PBS were transplanted into experimentally induced ischemic rats using NOD / SCID (severe combined immunodeficiency) mice. Laser Doppler perfusion image analysis showed that blood perfusion was significantly lowered on the hind legs injected with C-CD31 cells on day 21 (see Figures 3A and 3B).

High-performance microscopy revealed that the density of capillaries in terms of angiogenic blood vessels was much lower in the C-CD31 group than in the H-CD31 group in cell-injected hindlimb adducts.

Immunohistochemical methods were also used to measure the ability of the C-CD31 and H-CD31 to engraft or survive, and 1X106 dil-labeled cells were implanted in the hindlimb ischemic area at 4 weeks post-transplant. As a result, C-CD31 (7.5 + -4.4) showed a remarkably lowered graft performance than H-CD31 (21.7 + - 8.5) (see FIGS. 3E and 3F).

Example 5. Correlation Analysis between CAD and CD31 + Cell Count

To determine the number of CD31-expressing cells in peripheral blood, the number of CD31 + cells was measured. Interestingly, CD31 + cell counts were measured at a significantly higher level in UA patients than SA patients and controls (see Figures 4B and 4C).

In addition, the number of CD31 + cells was analyzed to be associated with coronary artery related disease (r = 0.362, P = 0.002) and cardiovascular risk factors (r = 0.514, P <0.001) (see FIGS. 4B and 4C).

Example 6. CAD prediction due to CD31 + cell number

The diagnostic value according to the number of CD31 + cells was determined as the mean value in the ROC curve (see FIG. 5). The diagnostic category of variable angina was 0.804 area in the AUC curve (P <0.0001). The CD31 + cell cycle number (≥ 18.5%) was the optimal cut-off value for UA prediction (sensitivity = 68.0%, specificity = 86.4%). Positive and negative predictive values were 85.0% (95% Cl 65.1 to 95.7%) and 70.4% (95% Cl 61.4 to 97.0%), respectively.

Example 7. Abundant inflammation-associated genes in CD31 + clusters

Real-time RT-PCR was performed to analyze expression patterns between CD31 + and CD31- cells. Expression patterns of inflammation-related genes were compared. As quantitative data, genes associated with major inflammatory or inflammatory signaling pathways such as IL-l [alpha], IL-6, IL-8, and TNF-y are significantly more abundant in peripheral blood-derived CD31 + (See FIG. 6). These results suggest that CD31 + cell cycle and inflammation-related cells are related.

Claims (7)

A composition for the diagnosis of coronary artery disease (CAD) comprising CD31 or fragments thereof. The composition for diagnosing coronary artery diseases according to claim 1, wherein the coronary artery disease is angina pectoris or myocardial infarction. (a) immobilizing the biological sample and the control sample to a fixture;
(b) treating the solid body with an antibody that specifically binds to CD31 or a fragment thereof;
(c) treating a marker capable of detecting antigen-antibody binding of step (b); And
(d) comparing the degree of labeling of the markers of step (c) in the biological sample and the control;
Lt; RTI ID = 0.0 &gt; CD31 &lt; / RTI &gt; or fragments thereof. The method according to claim 3, wherein the biological sample is at least one sample selected from the group consisting of tissue, cells, blood, serum, saliva, urine, and feces. 5. The method according to claim 4, wherein the blood is peripheral blood. 4. The method according to claim 3, wherein, in step (d), the level of expression of CD31 or fragment thereof in the biological sample is higher than that of the control, the CD31 or fragments thereof are predicted to have coronary artery disease or have coronary artery disease / RTI &gt; A kit for diagnosing coronary artery disease comprising an antibody or an aptamer that specifically binds to CD31 or a fragment thereof.

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