CN1928111B - Correlation analysis of circulation endothelium progenitor cell and coronary artery pathological changes - Google Patents

Abstract

The present invention discloses the correlation between circulating endothelial progenitor cells (EPC) and coronary artery lesions, especially the degree of coronary artery stenosis. The experiment of the present invention proves that there is a high correlation between the number of circulating endothelial progenitor cells and the ability of migration and adhesion with the degree of coronary artery stenosis, so it can be used as an auxiliary reference index for diagnosing the degree of coronary artery stenosis. The present invention also provides a method and a kit for detecting coronary artery lesions based on the correlation.

Description

Correlation Analysis of Circulating Endothelial Progenitor Cells and Coronary Artery Lesions

technical field

The present invention relates to the field of molecular biology. More specifically it relates to the correlation between circulating endothelial progenitor cells and coronary artery disease, especially the degree of coronary artery stenosis. The invention also relates to a method and a kit for detecting coronary artery lesions based on the correlation.

Background technique

Coronary artery disease is a disease that seriously endangers people's health.

Since Asahara et al. first successfully induced the differentiation of human peripheral blood CD34 ⁺ cells into endothelial cells in vitro in 1997 (Asahara T, Murohara T, Sullivan A. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science1997; 275: 964-7 ), more and more evidence shows that there are endothelial progenitor cells (EPCs) in adult bone marrow. When blood vessels are damaged, bone marrow EPCs are mobilized to peripheral blood, differentiate and integrate to the damaged site, and participate in the process of vascular endothelial repair.

Intact vascular intima plays an important role in maintaining vascular tension and vascular homeostasis. A variety of risk factors cause endothelial damage through oxidative stimulation, and then cause atherosclerosis (Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation2002; 105: 1135-43).

In recent years, it has been reported that circulating EPCs can repair damaged intima (Shi Q, Raffi S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cell. Blood1998; 92: 362-7). However, although some people speculate that the number of EPCs has a certain correlation with endothelial cell function and cardiovascular risk factors, before the present invention, the correlation between the number and function of EPCs and coronary artery lesions has never been confirmed.

To sum up, in order to prevent and treat coronary artery disease as early as possible, there is an urgent need in this field to find related factors of coronary artery disease, and to develop methods, kits, and related therapeutic drugs for detecting coronary artery disease.

Contents of the invention

The object of the present invention is to provide a method and detection kit for auxiliary diagnosis (especially early diagnosis) of coronary artery disease.

In a first aspect of the present invention, a method for screening candidate therapeutic agents for the treatment of coronary artery disease is provided, comprising the steps of:

(a) In the test group, adding the candidate to be screened to the culture of circulating endothelial progenitor cells, culturing and detecting the number of circulating endothelial progenitor cells; and, in the control group, without adding the candidate to be screened In some cases, culture and measure the number of circulating endothelial progenitor cells;

(b) comparing the number of circulating endothelial progenitor cells in the test group of step (a) to the number of circulating endothelial progenitor cells in the control group to which no candidate was added, if the number of circulating endothelial progenitor cells in the test group is statistically significant Higher than the control group, it indicates that the candidate is a candidate therapeutic agent for the treatment of coronary artery lesions.

In another preferred example, the method also includes the steps of:

(b') Detecting the migratory ability of circulating EPCs in the test group and comparing it with the migratory ability of circulating EPCs in the control group, wherein the migratory ability of the circulating EPCs in the test group was statistically significantly higher than that of the control group, indicating that the candidate is a candidate therapeutic agent for the treatment of coronary artery lesions.

In another preferred example, the method also includes the steps of:

(b") Detecting the adhesion ability of circulating endothelial progenitor cells in the test group and comparing it with the adhesion ability of circulating endothelial progenitor cells in the control group, wherein the adhesion ability of circulating endothelial progenitor cells in the test group is statistically significant Higher than the control group, it indicates that the candidate is a candidate therapeutic agent for the treatment of coronary artery lesions.

In another preferred example, the coronary artery lesion is coronary artery stenosis.

The present invention also includes the candidate therapeutic agent obtained by screening with the above method.

In the second aspect of the present invention, a use of a specific marker of circulating endothelial progenitor cells is provided for preparing reagents or kits for detecting coronary artery lesions. Preferably, the specific marker is selected from: CD133, KDR or vWF.

In another preferred example, the coronary artery lesion is coronary artery stenosis.

In the third aspect of the present invention, the use of an accelerator for promoting the number of circulating endothelial progenitor cells is provided for preparing a medicine for treating coronary artery disease.

In another preferred example, the coronary artery lesion is coronary artery stenosis.

In a fourth aspect of the present invention, a method for detecting coronary artery stenosis (or susceptibility) is provided, which includes the steps of: detecting the number of circulating endothelial progenitor cells in the individual to be tested, and comparing it with the circulating endothelial progenitor cells of the normal population (normal control) Compared with the number of endothelial progenitor cells, if the number of circulating endothelial progenitor cells in the individual to be tested is less than 30% (preferably less than 50%, more preferably less than 70%), it indicates that the individual has a high possibility of coronary artery stenosis in normal population.

Description of drawings

Figure 1 shows the relationship between the number of CD133/KDR positive cells, serum CRP concentration and coronary artery lesions.

Figure 2 shows the relationship between the number of CD133/KDR positive cells, serum CRP concentration and Gensini score

Figure 3 shows the phenotypic identification of EPCs.

Figure 4 shows the relationship between EPCs migration ability and coronary artery lesions.

Figure 5 shows the relationship between EPCs adhesion ability and coronary artery lesions.

Detailed ways

Through extensive and in-depth research, the inventors first discovered and confirmed the correlation between circulating endothelial progenitor cells (EPC) and coronary artery lesions (especially the degree of coronary artery stenosis). There is a high correlation between the number of circulating endothelial progenitor cells and the degree of coronary artery stenosis, so it can be used as an auxiliary reference index for diagnosing the degree of coronary artery stenosis. The invention provides a method and a kit for detecting coronary artery lesions based on the correlation.

Specifically, the present inventors analyzed the severity and risk factors of 104 patients with coronary angiography (excluding acute coronary syndrome and myocardial infarction); CD133/KDR was used as the EPCs marker, and the patients were detected by flow cytometry. The number of CD133/KDR double-labeled cells; Peripheral blood mononuclear cells were differentiated and cultured in 93 patients to detect the ability of EPCs migration and adhesion.

The test results showed that the number of EPCs in patients with positive coronary angiography was significantly lower than that in patients with negative coronary angiography (p<0.01); the number of EPCs was negatively correlated with Gensini score (n=49, r=-0.318, p=0.016). The number of peripheral blood EPCs was negatively correlated with age and hypersensitive C-reactive protein (hs-CRP) (p=0.001, 0.012); the number of EPCs was significantly reduced in patients with hypertension and family history of coronary heart disease (p=0.042, 0.043). The migration ability of EPCs in patients with coronary heart disease was also significantly lower than that in normal people, and the decline in multi-vessel disease was more significant than that in single-vessel disease (p<0.05). EPCs migration ability was negatively correlated with Gensini score (n=55, r=-0.315, p=0.021). The adhesion ability of EPCs in patients with coronary heart disease was significantly lower than that in normal people (p<0.05).

This shows that in patients with stable coronary heart disease, the number, migration and adhesion of circulating EPCs are lower than those of normal people, and the number and migration ability are related to the degree of coronary artery disease. The number of EPCs was correlated with cardiovascular risk factors and negatively correlated with serum CRP concentration. Since circulating EPCs have the effect of vascular repair, the angiogenesis ability of patients with coronary heart disease is weakened with the aggravation of coronary artery stenosis. Various cardiovascular risk factors, especially CRP, may promote the occurrence and development of atherosclerosis by affecting the number and function of EPCs. .

As used herein, the term "EPC" refers to circulating endothelial progenitor cells.

As used herein, the term "CRP" refers to C-reactive protein, which is a marker of inflammation.

Based on the new discovery of the present invention, the circulating endothelial progenitor cells have many new uses. These uses include (but are not limited to): direct use as a drug to treat coronary artery disease, especially coronary artery stenosis. In addition, substances that promote the number of circulating endothelial progenitor cells can also be screened, and these initially screened substances can form a screening library, so that people can finally screen out drugs that can effectively treat coronary artery lesions (especially coronary artery stenosis).

On the other hand, the specific markers of circulating endothelial progenitor cells can also be used in the present invention, by detecting circulating endothelial progenitor cells with these specific markers (things) in an individual, the number of circulating endothelial progenitor cells can be determined, thereby as an early Auxiliary index for auxiliary diagnosis of coronary artery disease (especially coronary artery stenosis).

Using circulating endothelial progenitor cells, substances that interact with circulating endothelial progenitor cells, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

An agonist or promoter that increases the number of circulating endothelial progenitor cells, etc., can increase the number of circulating endothelial progenitor cells when administered (administered) therapeutically, thereby improving the symptoms of coronary artery disease (especially coronary artery stenosis). Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, or subcutaneous administration.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of a promoter for promoting the number of circulating endothelial progenitor cells and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. The active ingredient is administered in a therapeutically effective amount, for example about 0.1 microgram/kg body weight to about 10 mg/kg body weight per day.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the number level of circulating endothelial progenitor cells. These test results can be used to assist in judging or diagnosing coronary artery disease (especially coronary artery stenosis).

A method for detecting the number of circulating endothelial progenitor cells is to use specific markers of circulating endothelial progenitor cells for detection, which includes: contacting the sample with specific antibodies against specific markers of circulating endothelial progenitor cells; observing whether the antibody complex is formed The formation of antibody complexes indicates the presence of circulating endothelial progenitor cells in the sample. Combined with equipment such as flow cytometry, the number of cells with this specific marker (ie, circulating endothelial progenitor cells) can be determined.

The main advantages of the present invention are:

For the first time, the correlation between circulating endothelial progenitor cells (EPCs) and coronary artery lesions (especially coronary artery stenosis) has been confirmed, so it is helpful for the diagnosis (especially early auxiliary diagnosis) and treatment of coronary artery lesions (especially coronary artery stenosis) A new way is provided.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1

method

1. Research object

From March to May 2004, 104 inpatients who underwent coronary angiography (CAG) were studied (CAG positive or negative). Patients with malignant tumors, peripheral vascular disease, and proliferative retinopathy were excluded; patients with acute coronary syndrome, acute myocardial infarction, inflammation, acute bleeding or blood transfusion history, and patients taking statins or erythropoietin within the past 2 months were excluded; Premenopausal women were excluded.

2. General clinical information

Collect basic information of all patients: gender, age, height, weight, blood pressure. Detailed medical history: hypertension, diabetes, coronary heart disease, cerebrovascular disease, kidney disease, smoking history, drinking history, lifestyle (whether to exercise), family history of coronary heart disease. And make routine laboratory tests: fasting and postprandial blood glucose, total cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglycerides, creatinine, uric acid.

3. Echocardiography

Two-dimensional, M-mode and Doppler echocardiography were performed on the patient with the M2410A-M2408A color Doppler ultrasonography instrument from Hewlett Packard Company of the United States, and the probe frequency was 2.5MHz. Follow the recommendations of the American Society of Echocardiography.

The left ventricular myocardial weight (LVM) was calculated according to Devereux formula, LVM(g)=1.04[(LVDd+IVST+PWT) ³ -LVDd ³ ]-13.6.

Body surface area (BSA) = 0.0061 x height (cm) + 0.0128 x body weight (kg) - 0.1529.

Left ventricular myocardial mass index (LVMI) adopts the formula: LVMI (g/m ² )=LVM/BSA (body surface area).

4. Coronary angiography

Selective coronary angiography was performed by Judkins method. Quantitative coronary angiography (QCA) was used to evaluate the vessels and stenosis of coronary artery lesions. Vascular stenosis ≥ 50% was used as a positive standard. A lesion involving one of the left anterior descending artery (LAD), left circumflex artery (LCX), and right coronary artery (RCA) is considered a single-vessel lesion, and those involving more than two vessels are considered multi-vessel lesions. Lesions of the left main coronary artery were two-vessel lesions. The severity of coronary artery lesions was evaluated in two ways: 1. The number of coronary arteries involved by lesions (divided into single-vessel lesions, two-vessel lesions and three-vessel lesions); 2. Gensini score (performed according to the method described in the following literature: Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol 1983; 51: 606).

5. Determination of serum hs-CRP, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1)

Serum hs-CRP concentration was determined by immunoscattering turbidimetry using a hypersensitive CRP kit (Dade Behring Inc. Marburg, Germany). The sensitivity is 0.175mg/L. ICAM-1 and VCAM-1 serum concentrations were determined with sICAM-1 and sVCAM-1 ELISA kits (DIACLONE, Besancon, France). The sensitivities were 8 ng/ml and 0.6 ng/ml.

6. Measurement of the number of circulating EPCs by flow cytometry

Before coronary angiography, Judkins catheter was inserted into the femoral artery to take 150ul of blood, and PE-labeled CD133 mouse anti-human monoclonal IgG1 antibody (MACS, Bergisch Gladbach, Germany), APC-labeled mouse anti-human KDR monoclonal IgG1 antibody (R&D, Minneapolis, USA). Isotype controls were PE-labeled and APC-labeled mouse IgG1 (Becton Dickinson, Franklin Lakes, USA). BD company FACSCalibur flow cytometer and CellQuest software were used for flow cytometric analysis. A window was set up for the lymphocyte population on the two-parameter point diagram of forward angle scattered light and side angle scattered light, and the number of collected cells was 60,000 in total, and the percentage of KDR/CD133 cells was counted based on this population.

7. Isolation and cultivation of EPC

Take 10ml of fasting peripheral blood, and obtain mononuclear cells by density gradient centrifugation within 4 hours. Mononuclear cells were seeded on fibronectin (FN)-coated culture plates (BD Biosciences, CA, USA). EGM medium (containing 10% fetal bovine serum, penicillin 100U/ml, streptomycin 100U/ml) (Cambrex, Walkersville, MD) was cultured for 4 days, non-adherent cells were washed off with PBS, and the culture medium was changed to continue to culture for 7 days , cytochemical analysis of adherent cells.

8. Identification of EPCs

Adherent cells were treated with 0.125% trypsin, and cells were labeled with PE-labeled anti-human vascular endothelial growth factor (KDR) monoclonal antibody (R&D, Minneapolis, USA) and Von Willebrand factor (vWF) antibody (DAKO, Glostrup, Denmark) , for flow cytometric analysis.

9. Detection of EPCs Migration Ability

Adherent cells were harvested and counted as described above. Mix 600 μl culture medium and vascular endothelial growth factor

(VEGF, 50ng/ml) was placed in the lower chamber of a transwell (8-μm pore size) (Corning Costar Inc., Corning, New York, USA), and 2×10 ⁴ EPCs were suspended in 200 μl of culture solution and injected into the upper chamber, and incubated for 24 hours. The unmoved cells on the filter membrane were scraped off, fixed with 75% ice ethanol, stained with HE, and 5 microscope fields (×200) were randomly selected to count the cells that migrated to the lower layer.

10. Detection of adhesion ability of EPCs

The adherent cells were collected by digestion with 0.25% trypsin, suspended in EGM medium, and counted. 1×10 ⁵ EPCs were spread on a FN-coated culture plate, incubated at 37°C for 30 min, and the adherent cells were counted.

11. Statistical analysis:

All data are presented as mean ± standard deviation. SPSS statistical software was used for data processing. CRP concentrations were converted to natural logarithm to achieve a normal distribution. The influencing factors of CD133/KDR ⁺ cells were analyzed by multivariate stepwise regression method. Partial regression analysis and covariance analysis were used to determine the relationship between the number of CD133/KDR ⁺ cells, EPCs migration, adhesion ability and severity of coronary artery lesions.

result

1. Clinical data of selected patients

80 patients with suspected or clinically confirmed coronary heart disease were selected as subjects. According to the results of coronary angiography, they were divided into three groups: negative coronary angiography, single-vessel disease and multi-vessel disease. There were no significant differences in age, body mass index, hypertension, smoking, alcohol drinking, family history of coronary heart disease, fasting blood glucose and blood lipid levels among the three groups, but males (p<0.01), history of diabetes (p<0.05) and alcohol consumption in the coronary heart disease group were significantly different. Those (p<0.05) were significantly increased; serum creatinine clearance was significantly reduced (p<0.01) (see Table 1).

Table 1. Clinical data of selected patients

Total CAG(-) single vessel disease Polyvascular disease P value N 104 44 35 25 age) 63.9±10.6 61.5±9.98 63.03±10.01 64.60±10.48 0.23 BMI(Kg/m ² ) 24.69±3.67 23.80±3.24 25.07±3.96 25.80±3.80 0.11 Gender (male, %) 77 (74.0%) 25 (57%) 29 (82%) 23 (92%) ＜0.01 Medical history, n(%) high blood pressure 55 (52.9%) 18 (45%) 20 (57.1%) 17 (60%) 0.21 diabetes 12 (11.5%) 1 (2.3%) 6 (17.1%) 5 (20.0%) 0.049 Current smoker 37 (35.6%) 16 (36.3%) 13 (37.1%) 8 (40%) 0.87 drinker 10 (9.6%) 2 (4.5%) 6 (17.1%) 2 (24%) 0.02 CAD family history 27 (26.0%) 9 (20.5%) 13 (37.1%) 5 (20%) 0.37 LVMI (g/cm ² )  119.33±28.78  114.3±25.46 120.43±20.81  126.64±26.62 0.51 blood test

Fasting plasma glucose (mmol/L) 5.75±1.11 5.49±0.68 5.84±1.27 6.07±1.40 0.15 Serum cholesterol (mmol/L) 4.81±1.05 4.81±0.90 4.80±1.22 4.82±0.85 0.89   Serum triglycerides (mmol/L) 1.71±0.90 1.66±0.90 1.73±1.11 1.76±0.53 0.88   Serum HDL-cholesterol (mmol/L) 1.26±0.41 1.36±0.49 1.18±0.31 1.18±0.34 0.39   Serum LDL-cholesterol (mmol/L) 2.79±0.78 2.77±0.70 2.81±0.94 2.82±0.70 0.92 Urea (umol/L) 346.27±85.65 334.35±75.14 348.11±84.01 364.69±103.61 0.65 Ccr (ml/min/1.73m ² ) 76.55±21.32 83.53±22.27 75.74±17.72 65.40±19.96 0.03

CAD: coronary artery disease;

BMI: body mass index;

LVMI: left ventricular mass index (left ventricular mass index);

CAG(-): normal coronary angiography (normal coronary angiography);

Ccr: creatinine clearance.

2. The relationship between peripheral blood CD133/KDR ⁺ cells and risk factors

The predictive effect of various risk factors on EPCs was analyzed by multiple regression method. Age, hs-CRP, hypertension, and family history of coronary heart disease were found to be associated with the number of EPCs (see Table 2).

Table 2 Relationship between cardiovascular risk factors and the number of circulating EPCs

standard regression coefficient (SE) P value age -0.390 (0.001) 0.001 Ln hs-CRP -0.2291 (0.003) 0.012 high blood pressure -0.166 (0.005) 0.042 CAD family history -0.170 (0.005) 0.043

hs-CRP: high sensitivity C active protein (C reactive protein);

ln: natural logarithm;

CAD: coronary artery disease;

SE: standard deviation

3. The relationship between the number of CD133/KDR ⁺ cells, hs-CRP and the degree of coronary artery disease

The patients were divided into three groups according to the number of coronary arteries involved by lesions, adjusted for age, sex and body surface area, and analyzed for covariance. The number of EPCs in the negative coronary angiography group was (0.064±0.004%)% (n=44); the single-vessel disease group was (0.043±0.004%)% (n=35); the multi-vessel disease group was (0.030±0.005%) (n=25). Compared with the negative coronary angiography group, the number of EPCs in the single-vessel lesion and multi-vessel lesion groups was significantly reduced (p<0.05; p<0.01), and the number of EPCs in the multi-vessel lesion group was also significantly lower than that in the single-vessel lesion group (p<0.05) . There were also significant differences in serum CRP concentrations among the three groups. In the negative coronary angiography group, ln CRP was 0.077±0.015 (n=44); in the single-vessel disease group, it was 0.725±0.125 (n=35); in the multi-vessel disease group, it was 1.343±0.206 (n=25) (Figure 1).

Corrected by age, sex and body surface area, partial regression analysis was performed on the number of CD133/KDR ⁺ cells, hs-CRP and Gensini score in patients with positive coronary angiography, and it was found that the number of CD133/KDR ⁺ cells and Gensini score were negatively correlated (n= 60, r=-0.355, P=0.006), hs-CRP was positively correlated with Gensini score (n=49, r=0.476, P=0.001) (Figure 2).

4. Phenotype identification of EPCs

Peripheral blood mononuclear cells were cultured adherently for seven days, and the cells were found to be spindle-shaped. The cells were identified by flow cytometry, and it was found that the cells expressed endothelial markers vWF (100%) and KDR (96%) ( FIG. 3 ).

5. The relationship between EPCs migration ability and coronary artery lesions

The ability of EPCs to migrate to VEGF plays an important role in the process of angiogenesis. By detecting the migration ability of circulating EPCs in 38 patients with negative coronary angiography and 55 patients with coronary heart disease, it was found that the migration ability of EPCs in CAG(-) patients (EPCs migration 38.73±1.37/*200, n=38) was significantly higher than that in single vessels Lesions (EPCs migration 34.33±1.58/*200, n=27) and multivessel disease (EPCs migration 29.46±1.66/*200, n=27) and EPCs migration ability was inversely proportional to Gensini score (n=55, r= -0.315, p=0.021) (Figure 4).

6. The relationship between the adhesion ability of EPCs and coronary artery lesions

The adhesion ability of EPCs was significantly decreased in patients with positive coronary angiography (p<0.05), but there was no correlation between adhesion ability and Gensini score (n=55, r=-0.196, p=0.159) (Figure 5)

discuss

The present invention measures the number and functional activity of circulating EPCs in patients with stable coronary heart disease, and finds that the number, migration ability, and adhesion ability of EPCs in patients with stable coronary heart disease are lower than those of normal people; for the first time, it is found that the number and migration function of EPCs are related to the degree of coronary artery stenosis ; And for the first time in a clinical study found that the number of circulating EPCs and serum hs-CRP levels were negatively correlated.

Using CD133/KDR as the EPCs marker to detect circulating EPCs in patients, it was found that the number of circulating EPCs in patients with positive coronary angiography was significantly lower than that in patients with negative coronary angiography; Angiographic Gensini score was negatively correlated. The above results can be inferred: in patients with severe coronary artery disease, the production and mobilization of EPCs are reduced, and the half-life is shortened.

The number of circulating EPCs is very small. When blood vessels are injured, cytokines, such as VEGF and SDF-1, are released locally, which have a chemotactic effect on EPCs and enable them to play a role in the ischemic site. Therefore, in addition to the number of EPCs, the migration function of EPCs plays a key role in the process of angiogenesis, and VEGF is considered to be the most important inducer. In the present invention, VEGF is used as an inducing factor to detect the migration function of circulating EPCs, and it is found that there are significant differences in the migration ability of EPCs in the CAG-negative group, the single-vessel disease group and the multi-vessel disease group, and the migration ability is negatively correlated with the Gensini score. It suggested that the migration ability of circulating EPCs decreased with the aggravation of coronary artery disease. VEGF induces endothelial cell migration to participate in angiogenesis through VEGFR-2 (KDR), thus suggesting that EPCs may also participate in this process through KDR. However, the present invention does not find that KDR expression is related to coronary artery disease, so there may be a downstream signal transduction pathway that reduces the migration ability of EPCs.

Another important mechanism by which EPCs participate in angiogenesis is cell adhesion. EPCs integrate into endothelial injury sites through cell-cell adhesion and cell-extracellular matrix adhesion to repair blood vessels. The present invention finds that the adhesion function of EPCs to FN in patients with coronary heart disease is significantly reduced, but no correlation between the adhesion function and the severity of coronary artery disease is found.

Changes in the number and biological function of EPCs in coronary heart disease may be mediated by the following mechanisms:

First of all, angiotensin II and reactive oxygen species (ROS) are substances that play an important role in the pathogenesis of coronary artery lesions. It has been reported in the literature that angiotensin II promotes aging and apoptosis of EPCs by inducing oxidative stimulation (Imanishi T, Hano T, Nishio I .Angiotensin II accelerates endothelial progenitor cell senescence through induction of oxidative stress. JHypertens 2005; 23:97-104.).

Secondly, eNOs can accelerate the mobilization of EPCs, and the function of nitric oxide synthase (eNOs) produced by vascular endothelium in patients with coronary heart disease is impaired, thereby reducing the number of EPCs. For patients with coronary heart disease, hypoxia-induced reduction in VEGF release may also be an important factor in reducing the mobilization, differentiation, and migration of EPCs. In addition, ox-LDL, which plays an important role in the process of atherosclerosis, can inhibit the differentiation of EPCs and affect the ability of EPCs to migrate and angiogenesis. On the other hand, chronic vascular endothelial injury associated with atherosclerosis can continuously consume circulating EPCs to participate in endothelial repair, thereby reducing the more functional EPCs in circulation. Cardiovascular risk factors, including the inflammatory factor CRP, may also play an important role in this process. The intact vascular intima maintains the normal function of blood vessels, and endothelial injury can cause atherosclerosis. Circulating EPCs are the "cell bank" for repairing damaged endothelium. Under normal circumstances, there is a dynamic balance between endothelial injury and EPCs repair to maintain intimal integrity, but the reduction in the number/function of EPCs may affect endothelial repair, resulting in damage to intimal integrity and promoting the occurrence and development of atherosclerosis. The present invention finds that the number of EPCs is significantly negatively correlated with the severity of coronary artery disease, suggesting that EPCs mobilized from bone marrow to peripheral blood is not enough to repair damaged endothelium is one of the pathogenesis of coronary heart disease.

The present invention finds that the number of CD133/KDR positive cells is negatively correlated with the serum CRP level. George et al. (George J, Goldstein E, Abashidze S, et al. Circulating endothelial progenitor cells in patients with unstable angina: association with systemic inflammation. Eur Heart J 2004; 25: 1003-8.) in coronary heart disease (including stable coronary heart disease and Acute coronary syndrome) EPCs in the study found that the number of EPCs and CRP concentration is positively correlated. Myocardial ischemia caused by acute coronary syndrome may induce the release of VEGF, thereby increasing the differentiation and mobilization of EPCs, and increasing the number of circulating EPCs; while CRP is an inflammatory index, which increases significantly during acute inflammation. Recent literature suggests that hs-CRP is not only an important cardiovascular risk factor, but also an important participant in atherosclerosis. In vitro experiments found that CRP significantly reduced the number of EPCs, delayed the appearance of endothelial-specific markers, promoted the apoptosis of EPCs, and impaired the angiogenesis ability of EPCs. Another experiment suggests that: CRP inhibits the angiogenesis ability of EPCs by reducing the secretion of various cytokines. For the first time, the inventors found a negative correlation between EPCs and CRP from clinical studies, further confirming that CRP can reduce the number of EPCs. Since CRP is an important predictor of cardiovascular events, and EPCs are related to CRP, the number of circulating EPCs can be a biological marker for predicting cardiovascular disease. The present invention also shows that the number of circulating EPCs is related to cardiovascular risk factors such as age, hypertension, and family history of coronary heart disease. Therefore, this suggests that both traditional risk factors and CRP can reduce the number of circulating EPCs, impede endothelial repair, and promote atherosclerotic lesions.

Since the number and function of circulating EPCs are related to the severity of coronary artery stenosis, that is, the degree of coronary artery stenosis is negatively correlated with the angiogenesis ability of EPCs, it suggests that in some coronary artery lesions, various factors including CRP, It may reduce the number and function of EPCs, hinder endothelial repair, and accelerate the occurrence and development of atherosclerosis.

Example 2

Screening for accelerators that boost EPC numbers

Take 10ml of fasting peripheral blood, and obtain mononuclear cells by density gradient centrifugation within 4 hours. The same number (about 1×10 ⁵ ) of mononuclear cells were seeded on 6 culture plates coated with fibronectin (FN) (BD Biosciences, CA, USA).

In the 3 culture plates of the control group, culture in EGM medium (containing 10% fetal bovine serum, penicillin 100U/ml, streptomycin 100U/ml) (Cambrex, Walkersville, MD) without adding any compound to be tested 4 days.

In the 3 culture plates of the control group, in the EGM medium (containing 10% fetal bovine serum, penicillin 100U/ml, streptomycin 100U/ml) (Cambrex, Walkersville, MD) with a concentration of 0.1M of the compound to be tested ) for 4 days.

After 4 days of culture, wash off the non-adherent cells in the test group and the control group with PBS, change the culture medium and continue to culture for 7 days, and conduct cytochemical analysis on the adherent cells respectively.

Adherent cells were treated with 0.125% trypsin, and cells were labeled with PE-labeled anti-human vascular endothelial growth factor (KDR) monoclonal antibody (R&D, Minneapolis, USA) and Von Willebrand factor (vWF) antibody (DAKO, Glostrup, Denmark) , for flow cytometric analysis.

A test compound is considered to be effective if the number of circulating EPCs (average of 3 culture plates) is 30% higher in the test group compared to the number of circulating EPCs in the control group (average of 3 culture plates). A more effective enhancer of EPC numbers; a test compound was considered to be a very effective enhancer of EPC numbers if the number of circulating EPCs (average of 3 culture plates) was 50% higher in the test group.

Example 3

Screening for compounds that promote the ability of circulating endothelial progenitor cells to migrate and adhere

For the promoters screened in Example 2 to promote the number of EPCs, it was further determined whether they have the ability to promote the migration and adhesion of circulating endothelial progenitor cells:

(a) Detection of migration ability of EPCs

Adherent cells were collected and counted as in Example 2. Put 600 μl of culture medium and vascular endothelial growth factor (VEGF, 50ng/ml) into the lower chamber of a transwell (8-μm pore size) (Corning Costar Inc., Corning, New York, USA), and suspend 2×10 ⁴ EPCs in 200 μl The culture solution was poured into the upper chamber, cultured for 24 hours, the unmoved cells on the filter membrane were scraped off, fixed with 75% ice ethanol, stained with HE, and 5 microscope fields (×200) were randomly selected to count the cells that migrated to the lower layer.

(b) Detection of adhesion ability of EPCs

The adherent cells were collected by digestion with 0.25% trypsin, suspended in EGM medium, and counted. 1×10 ⁵ EPCs were spread on a FN-coated culture plate, incubated at 37°C for 30 min, and the adherent cells were counted.

The results showed that none of the two promoters obtained from the preliminary screening to promote the number of EPCs could promote the migration and adhesion of circulating endothelial progenitor cells. This suggests that promoting EPC proliferation involves different pathways from migration and adhesion.

Example 4

Kit for detection of susceptibility to coronary artery stenosis using specific markers of circulating endothelial progenitor cells

Prepare a kit containing: (a) PE-labeled CD133 mouse anti-human monoclonal IgG1 antibody (MACS, Bergisch Gladbach, Germany) and (b) APC-labeled mouse anti-human KDR monoclonal IgG1 antibody (R&D, Minneapolis, USA).

For a test group consisting of 10 coronary artery stenosis patients and 10 normal volunteers, 150ul of blood was collected by inserting a Judkins catheter into the femoral artery. Under double-blind conditions, PE-labeled CD133 mouse anti-human monoclonal IgG1 antibody (MACS, Bergisch Gladbach, Germany), APC-labeled mouse anti-human KDR monoclonal IgG1 antibody (R&D, Minneapolis, USA) were added. Isotype controls were PE-labeled and APC-labeled mouse IgG1 (Becton Dickinson, Franklin Lakes, USA). BD company FACSCalibur flow cytometer and CellQuest software were used for flow cytometric analysis. A window was set up for the lymphocyte population on the double-parameter dot plot of forward angle scatter light and side angle scatter light, and a total of 50,000 cells were collected, and the percentage of KDR/CD133 double positive cells was counted in this population. Coronary angiography was performed on all test subjects at the same time.

Judgment criteria:

(a) The percentage of KDR/CD133 double-positive cells > 0.05%, indicating that there is no coronary artery stenosis;

(b) The percentage of KDR/CD133 double-positive cells is less than 0.045%, indicating that coronary artery stenosis or susceptibility to coronary artery stenosis is higher than normal population;

(c) The percentage of KDR/CD133 double positive cells is between 0.045% and 0.05%, which has no definite diagnostic value.

result:

The percentage of KDR/CD133 double-positive cells in 8 testers was <0.045%, and 7 of them had coronary artery stenosis (single-vessel disease or multi-vessel disease).

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (2)

1. A method for screening a candidate therapeutic agent for the treatment of coronary artery disease, characterized in that it comprises steps: (a) In the test group, adding the candidate to be screened to the culture of circulating endothelial progenitor cells, culturing and detecting the number of circulating endothelial progenitor cells; and, in the control group, without adding the candidate to be screened In some cases, culture and measure the number of circulating endothelial progenitor cells; (b) comparing the number of circulating endothelial progenitor cells in the test group of step (a) to the number of circulating endothelial progenitor cells in the control group to which no candidate was added, if the number of circulating endothelial progenitor cells in the test group is statistically significant is higher than the control group, it indicates that the candidate is a candidate therapeutic agent for the treatment of coronary artery disease; And the method also includes: determining the effect of the candidate on the number of circulating endothelial progenitor cells in peripheral blood and the concentration of hypersensitive C-reactive protein in serum, and selecting the amount that causes the number of circulating endothelial progenitor cells in peripheral blood and the concentration of hypersensitive C-reactive protein in serum. The concentration of the reactive protein was inversely correlated with the candidate as a candidate therapeutic agent for the treatment of coronary artery lesions. 2. The method of claim 1, wherein the coronary artery lesion is coronary artery stenosis.

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