Recently, a number of studies have indicated the essential role of inflammatory factors, which are participated in the progression and formation of atherosclerosis, in the occurrence and development of coronary heart disease (CHD). The occurrence of acute coronary syndrome (ACS) is closely related to the presence of unstable plaques and secondary thrombosis. The inflammatory cells in plaques and their inflammatory products may be the cause for plaque instability and ruptures. Studies found three novel inflammatory markers, including lipoprotein-associated phospholipase A2 (Lp-PLA2), intracellular adhesion molecule-1 (ICAM-1), and chitinase-3-like protein 1 (YKL-40), which were involved in the clinical prognosis and pathogenesis of CHD. Nevertheless, the association between these three inflammatory markers and plaque stability and the severity degree of coronary artery stenosis need to be further studied.
In the study, enzyme-linked immunosorbent assay (ELISA) was applied to determine the levels of serum inflammatory factors ICAM-1, YKL-40, and Lp-PLA2 in CHD patients. This study aimed to explore the association between the inflammatory markers and CHD and its different clinical types, plaque stability and severity of coronary lesions, and the role of the inflammatory markers in the pathogenesis mechanism of ACSs, so as to build a reliable basis for clinical practice.
The study was approved by the Ethics Committee of the Beijing Anzhen Hospital Affiliated to Capital Medical University (No. 2015031) and signed informed consent was obtained.
In total, 120 patients aged 35–75 years and diagnosed with CHD by coronary arteriography from July 2015 to September 2016 in the Department of Cardiology, Beijing Anzhen Hospital Affiliated to Capital Medical University were included in this study. Sixty-nine cases were categorized into an ACS group and 51 cases into a stable angina pectoris (SAP) group. The CHD patients with ≥50% stenosis in at least one main coronary artery confirmed by coronary arteriography were enrolled. Twenty patients with chest pain symptom but normal coronary arteriograms were used as the control group. The 120 CHD patients were categorized into a single-vessel lesion group (45 cases), a double-vessel lesion group (38 cases), and a triple-vessel lesion group (37 cases) according to the number of lesion vessels. The Gensini score was used to evaluate the severity of coronary artery stenosis. The CHD patients were categorized into a mild lesion group (<26 scores, 36 cases), a moderate lesion group (26–54 scores, 48 cases), and a severe lesion group (>54 scores, 36 cases) according to the Gensini score.
Criteria for diagnosis
The ACS was diagnosed according to two guidelines. The first one is the American College of Cardiology Foundation and the American Heart Association Guideline, which was issued in 2013 and used for managing ST-elevation myocardial infarction. The second one is the “Non-ST elevation-ACS diagnosis guideline” which was issued in 2012 by the Chinese Society of Cardiology of Chinese Medical Association.
Criteria for inclusion and exclusion
Patients were included if they met the following criteria: (1) 35–75 years old; (2) being diagnosed with acute ACS; and (3) being subjected to coronary angiography within 3 days. All patients have signed the informed consent. The patients were excluded if they had uncontrolled grade 3 hypertension, severe valvular heart disease, or severe cardiac insufficiency (ejection fraction <35%); if they had hematopoietic system disorders, thyroid disorder, severe liver diseases, nervous system diseases, renal dysfunction, psychiatric illness, serious infectious diseases, or malignant tumor; if they had diabetes with glycated hemoglobin of 9.5% and random blood glucose of 13.7 mmol/L; if they were breastfeeding women or pregnant; and if they were involved in other clinical trials.
The patients were subjected to fasting elbow venous blood collection in the next morning after being admitted, followed by coronary angiography in 3 days. The following clinical data were recorded, including blood pressure, blood lipid, fasting blood glucose, liver and kidney functions, electrocardiograms, age, and gender.
Coronary stenosis assessment
The severity of coronary artery stenosis was assessed by quantitative coronary angiography using Gensini scores. The Gensini scores of 1, 2, 4, 8, 16, and 32 points were given for the stenosis of 1–25%, 26–50%, 51–75%, 76–90%, 91–99%, and 100% (complete occlusion). A factor was given to each segment of the coronary artery. The higher the factor, the more important the lesion's location is. The second diagonal branch was given a factor of 0.5; the first diagonal branch, the distal segment of anterior descending, the obtuse marginal branch, the proximal, middle, or distal segment of the right coronary artery, the posterior descending artery, the distal segment of circumflex artery, and left ventricular posterior branch were given a factor of 1; the mid segment of anterior descending was given a factor of 1.5; the proximal anterior descending or circumflex artery was given a factor of 2.5; and the left main lesion was given a factor of 5. The coronary artery stenosis was assessed by the multiplication of the factor of each segment and the corresponding score. The score of each coronary artery lesion was summed to obtain the Gensini score for each patient.
Measurement of inflammatory mediators
Five milliliter of fasting venous blood was drawn from each patient in the morning after being admitted for the measurement of biochemical indicators such as blood glucose and blood lipids. Another 5 ml of venous blood was drawn and centrifuged at 3000 r/min for 10 min. The supernatant serum layer was collected and then stored at −80°C. The levels of soluble ICAM-1, Lp-PLA2, and YKL-40 were measured by ELISA. For ICAM-1 and Lp-PLA2, the kit from R&D Systems Corporation (USA) was used, and for YKL-40, the kit from Quidel Corporation (USA) was used.
The statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were presented as mean ± standard deviation, and categorical variables were expressed as percentages. The mean values were compared using one-way analysis of variance, followed by post hoc tests including Mann–Whitney U-test and Kruskal–Wallis test; and the percentages were compared using Chi-square test. P < 0.05 was considered to be statistically significant.
There was no statistical difference in terms of age, gender, hypertension history, diabetes history, smoking history, admission blood pressure, fasting blood sugar (FBS), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) among the control group, the SAP group and the ACS group [all P > 0.05, Table 1]. There was no significant difference in terms of age, gender, hypertension history, smoking history, admission blood pressure, FBS, TG, TC, HDL-C, and LDL-C among the control group, the single-vessel lesion group, the double-vessel lesion group, and the triple-vessel lesion group [all P > 0.05, Table 2], but the number of patients with diabetes history in the double-vessel lesion group and triple-vessel lesion group were significantly more than that in the control group or the single-vessel lesion group [P < 0.05, or P < 0.01, Table 2]. The above indicators were not statistically different among the control group, the mild lesion group, the moderate lesion group, and the severe lesion group [all P > 0.05, Table 3].
Comparison of the concentration of inflammatory factors for patients with different types of coronary heart disease
The serum inflammatory factors ICAM-1, YKL-40, and Lp-PLA2 levels were significantly higher in the ACS group than those in control group and SAP group (P< 0.05 or P< 0.01 for each comparison); and compared with control group, no significant difference was observed in terms of the serum ICAM-1, YKL-40, and Lp-PLA2 levels in the SAP group [P > 0.05, Figure 1].
Comparison of the concentration of inflammatory factors for different numbers of coronary artery lesions
The serum ICAM-1, YKL-40, and Lp-PLA2 levels were no significant difference among control group, single-vessel disease group, double-vessel disease group, and three-vessel disease group [P > 0.05 for each comparison, Figure 2].
Comparison of the concentration of inflammatory factors for groups with different Gensini score
The serum ICAM-1, YKL-40, and Lp-PLA2 levels were no significant difference among control group, mild CHD group with its Gensini score <26, moderate CHD group with its Gensini score being 26–54, and severe CHD group with its Gensini score >54 [P > 0.05 for each comparison, Figure 3].
Correlation between inflammatory mediators and Gensini score
Nonparametric Spearman correlation analysis showed that serum ICAM-1, YKL-40, and Lp-PLA2 were not related the Gensini score in CHD patients (r = 0.093, r = –0.149, and r = –0.085, P > 0.05, respectively).
Inflammation plays an essential role in the development, progression, and prognosis of CHD, and has been widely concerned as an independent risk factor for the development of CHD. It is involved in the formation and progression of atherosclerosis, and the activation of inflammatory responses may be a major contributor to plaque instability. Ross et al. pointed out that atherosclerosis is a chronic inflammatory disease, and many inflammatory cells and factors participate in its occurrence and development process. The pathogenesis of the disease is as follows: vascular endothelial cells are damaged at the early stage of atherosclerosis, the endothelial cells adhere to monocytes with each other, and the monocytes adhere to the endothelial surface and pass through to the subcutaneous region to participate in the local inflammatory response. Under the stimulation of various substances, endothelial cells express a variety of pro-inflammatory molecules which play an important role in cell recruitment, migration, cell proliferation, and lipid and protein synthesis regulation together with nitrogen oxide and lipid mediators. Macrophages express the scavenger receptors under the action of oxidized low-density lipoprotein, which mediate the macrophages to swallow a large amount of lipid and concert to foam cells. The activated macrophages can secrete and mediate acute inflammatory reactions and can synthesize and secrete a variety of growth factors and stimulate the migration and proliferation of smooth muscle cells and fibroblasts. The activated T cells release cytokines such as interferon-γ and tumor necrosis factor-alpha. The activation of pro-inflammatory cells and the expression of cytokines promote the thickening or rupture of atherosclerotic plaques, and then the lipid pool enters the blood vessel to cause thrombosis, which then leads to the occurrence and development of ACS. ICAM-1, YKL-40, and Lp-PLA2 are newly discovered inflammatory markers in the pathogenesis and clinical prognosis of CHD. However, the relationship between these inflammatory markers and plaque stability and the severity degree of coronary artery stenosis need to be further studied.
ICAM-1 is mainly expressed on endothelial cells and immune cell membranes. It is a cell surface transmembrane glycoprotein and can mediate the intercellular interactions and the interactions between cells and extracellular matrix. As an essential biomarker of the activation of vascular endothelial cells, ICAM-1 can mediate the leukocyte-blood vessel wall adhesion, and the expression of ICAM-1 was found to be increased in plaques in CHD. In addition, the single nucleotide polymorphism rs281432 of ICAM-1 was found to be an independent factor for the prediction of the progression of CHD.
YKL-40 is expressed in smooth muscle cells, macrophages, endothelial cells, and several types of tumor cells. It is also an inflammatory glycoprotein involved in the formation of atherosclerotic plaques. The underlying mechanism might be that, as an endothelial dysfunction marker, YKL-40 can promote cell adhesion, chemotaxis/migration, and proliferation of vascular endothelial cells, resulting in neovascularization and vascular stenosis.
Nøjgaard et al. showed that the expression level of YKL-40 in the ACS group was significantly higher than that in the SAP group, but there was no significant difference between the control group and the SAP group. In a study involving 200 patients undergoing coronary angiography, Kucur et al. found that the expression level of YKL-40 increased with the increase of the number of coronary artery lesions. Zheng et al. indicated that YKL-40 was involved in the plaque injury of CHD and was a predictor of plaque injury. The level of YKL-40 was gradually increased with the increase of the number of coronary artery lesions and had a positive correlation with the Gensini score, and was a good predictor of plaque injury. However, it was suggested that there was no correlation between YKL-40 level and the severity of coronary artery lesions.
As an important biomarker for inflammation and vascular endothelial dysfunction, Lp-PLA2 was extensive studied in recent years. It is mainly secreted by smooth muscle cells, endothelial cells, and macrophages, and is regulated by mediators of inflammation. A number of studies disclosed the biological role of Lp-PLA2 in the formation and development of atherosclerosis plaques, and in the increase of the instability of the plaques, so that it was regarded as a new inflammatory response marker of atherosclerosis. Lp-PLA2 is not easy to be affected by rheumatic diseases, hypertension, blood pressure, body mass index, diabetes, infections, and other systemic factors. In addition, Lp-PLA2 is a more specific inflammatory marker, not such as fibrinogen, C-reactive protein, and other traditional non-specific ones, and exhibits a higher association with cardiovascular diseases. Furthermore, studies also discovered a number of other contributions of Lp-PLA2, including evaluation of the activity of coronary artery lesions, independent prediction of coronary events or other adverse cardiovascular events, and determination of the severity of CAD and identification of patients at high risk for risk stratification.
Our results showed that the levels of ICAM-1, YKL-40, and Lp-PLA2 in the ACS group were significantly higher than those in the SAP group and the control group, but there was no significant difference between the SAP group and the control group, indicating that ACS patients had more inflammation-related serum evidence reflecting the severity of the disease and plaque stability. Inflammation was involved in the development of atherosclerosis, especially promoted the formation of plaque instability, which is consistent with the previous findings. In addition, the study showed that the levels of ICAM-1, YKL-40, and Lp-PLA2 were not significantly different in the control group, the single-vessel lesion group, the double-vessel lesion group, and the triple-vessel lesion group. Therefore, the levels of ICAM-1, YKL-40, and Lp-PLA2 in the patients with coronary artery disease were not correlated with the range and severity of coronary artery stenosis. To further explore the correlation between ICAM-1, YKL-40, and Lp-PLA2 levels and the range and severity of coronary artery stenosis, we used a more accurate Gensini scoring system to quantitatively evaluate the severity of coronary artery stenosis of the main coronary artery. The results showed that there was no significant correlation between serum ICAM-1, YKL-40, and Lp-PLA2 levels and Gensini scores. Therefore, this study shows that serum ICAM-1, YKL-40, and Lp-PLA2 levels are better indicators for the plaque instability than the range of severity of coronary artery stenosis, which is different from previous studies and needs to be further validated.
This study suggests that the changes in serum inflammatory cytokines ICAM-1, YKL-40, and Lp-PLA2 are involved in the development of coronary atherosclerosis and are associated with ACSs and plaque stability, but are not significantly associated with the range of severity of coronary artery stenosis of CHD patients, suggesting that they are better indicative of the plaque instability than the severity of the disease of CHD patients, which provides new ideas for the prevention, diagnosis and treatment of CHD. However, the number of cases in this study is small, and there are many in vivo factors that affect the ICAM-1, YKL-40, Lp-PLA2, and YKL-40 levels so that the results of the study need to be further confirmed by large-size clinical studies.
Financial support and sponsorship
This work was supported by a grant from the National Basic Research Program of China (973 program, No. 2015CB554404).
Conflicts of interest
There are no conflicts of interest.
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Edited by: Li-Shao Guo