CLINICAL INVESTIGATION
Plasma Levels of C1q/TNF-Related Protein 1 and Interleukin 6 in Patients With Acute Coronary Syndrome or Stable Angina Pectoris Jun-Nan Tang, PhD, De-Liang Shen, PhD, Cong-Lin Liu, PhD, Xiao-Fang Wang, PhD, Li Zhang, MD, Xue-Xi Xuan, MD, Ling-Ling Cui, PhD and Jin-Ying Zhang, PhD
Abstract: Background: C1q/TNF-related protein 1 (CTRP-1), a novel adipocyte factor, may participate in the mechanisms of metabolism and inflammation. Interleukin 6 (IL-6) is a proinflammatory cytokine that is correlated with the severity of coronary heart disease (CHD). In this study, we focused on the levels of CTRP-1 and IL-6 in patients with CHD. Methods: Circulating CTRP-1 and IL-6 levels were measured using enzyme-linked immunosorbent assay in 81 patients with acute coronary syndrome (n 5 41) or stable angina pectoris (n 5 40). CTRP1 and IL-6 levels were also examined in 30 healthy individuals (control group). We examined the correlations between the levels of CTRP-1 and IL-6 and cardiac risk factors in CHD. Logistic regression analysis was performed to screen for factors that predict CHD. Results: Both CTRP-1 and IL-6 concentrations were increased in the acute coronary syndrome or stable angina pectoris group compared with the control group (P , 0.01). Both plasma levels of CTRP-1 and IL-6 in the single-, double- and triple-vessel lesion group were higher compared with the control group (P , 0.01). CTRP-1 levels were positively correlated with IL-6 (r 5 0.667, P , 0.01) and high-sensitivity C-reactive protein levels (r 5 0.520, P , 0.01) and negatively correlated with HDL-C (r 5 20.432, P , 0.01). The logistic regression analysis showed that increases in CTRP-1 and IL-6 levels may be powerful predictors of CHD. Conclusions: The variation of plasma CTRP-1 and IL-6 concentrations may play an important role in reflecting the degree of inflammation in CHD and the severity of coronary arterial atherosclerosis. This potential suggests that evaluating CTRP-1 and IL-6 in combination may aid in predicting the occurrence of CHD. Key Indexing Terms: C1q/TNF-related protein 1; Interleukin 6; Coronary heart disease; Acute coronary syndrome; Stable angina pectoris. [Am J Med Sci 2015;349(2):130–136.]
C
oronary heart disease (CHD) is one of the major cardiovascular diseases, which has seriously impacted human health globally. CHD is an inflammatory disease in which immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial tree.1 As a vital endocrine organ, adipose tissue secretes various types of bioactive substances, known as adipokines, that play important roles in the pathogenesis of obesity, From the Department of Cardiology (J-NT, D-LS, C-LL, X-FW, LZ, X-XX, J-YZ), the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; Institute of Clinical Medicine (J-NT), the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; and College of Public Health (L-LC), Zhengzhou University, Zhengzhou, China. Submitted May 20, 2014; accepted in revised form September 25, 2014. Supported by the Science and Technology Talents Team Construction Program of Zhengzhou City-Science and Technology Talents (131PLJRC670) and the Science and Technology Research Projects of Henan Province (13A320623). The authors have no conflicts of interest to disclose. Correspondence: Jin-Ying Zhang, PhD, Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China (E-mail: [email protected]).
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diabetes, hypertension and cardiovascular diseases.2 Recently, a new protein family secreted by adipokines, C1q/TNF-related protein (CTRP), was cloned on the basis of its sequence homology with adiponectin.3 This family of adiponectin paralogs may be involved in energy homeostasis and obesity-related inflammation.4 C1q/TNF-related protein 1 (CTRP-1), a member of the CTRP family, is secreted by stromal vascular cells (SVCs) composed of adipose-tissue macrophages, preadipocytes and endothelial cells.5 In human tissues, CTRP-1 mRNA is expressed more highly in the heart than in many other tissues such as the liver and kidney.6 Increased CTRP-1 gene expression can be indirectly induced by lipopolysaccharide (LPS) in epididymal adipose tissue and is regulated by the inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) in association with a decrease in adiponectin mRNA expression,7 indicating that adipose tissue CTRP-1 expression is induced in inflammation. In addition, Lasser et al8 demonstrated that CTRP-1 could specifically bind to fibrillar collagen type I and block collagen-induced platelet activation and aggregation. These observations indicate that CTRP-1 may play a role in the process of plaque formation in CHD. Interleukin 6 (IL-6) is a proinflammatory cytokine that is triggered by vulnerable plaque or necrotic myocardium and is correlated with the severity of coronary artery disease (CAD).9 In patients with CAD, elevated circulating IL-6 concentrations may be involved in the function of macrophage/foam cells present in atheromatous plaques.10 At present, data are limited regarding the correlation between plasma concentrations of CTRP-1 and IL-6 and the severity of CHD. Therefore, in this study, we measured plasma concentrations of these biomarkers and investigated their relationship with disease severity in patients with CHD.
METHODS Study Participants and Definition of Coronary Heart Disease The study was approved by the Institution Review Board of the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from every participant. From May 1, 2013, to February 1, 2014, 81 consecutive patients with CHD (51 men; mean age: 56.48 6 10.02 years; range, 39–85) were recruited from the Department of Cardiology in the First Affiliated Hospital of Zhengzhou University. All patients had CHD confirmed by coronary angiography (CAG). The enrolled patients were classified into 2 groups: a group with acute coronary syndrome (ACS) and a group with stable angina pectoris (SAP). The ACS group consisted of 20 patients with ST-elevation acute myocardial infarction and 21 patients with unstable angina pectoris (UAP). The SAP group consisted of 40 patients with SAP. Percutaneous coronary
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intervention was performed in 81 patients with ACS and SAP. During the same period, 30 healthy subjects (18 men; mean age: 50.87 6 10.56 years; range, 34–77) were recruited as a control group, excluding individuals with a history of CVD (myocardial infarction, unstable angina, stroke or cardiovascular revascularization), type 2 diabetes, stage 2 hypertension (resting blood pressure $160/100 mm Hg), malignancy or severe renal or hepatic disease. Acute myocardial infarction (AMI) was defined as a typical increase and gradual decrease of biochemical markers of myocardial necrosis (detection of a rise and/or fall of cardiac biomarkers such as CK-MB and/or troponin-T with at least 1 value above the 99th percentile upper reference limit) and at least 1 of the following: Ischemic symptoms, electrocardiogram changes indicative of new ischemia (new ST-T changes or new left bundle branch block), development of pathologic Q waves on electrocardiogram and imaging evidence of a new loss of viable myocardium or new regional wall motion abnormality.11 The diagnostic criteria for UAP included chest pain at rest with either a ST segment depression of at least 0.1 mV or a T-wave inversion in 2 or more continuous electrocardiographic leads and no biomarkers of myocardial necrosis (based on 2 or more blood samples collected at least 6 hours apart, with a reference limit of the 99th percentile of the normal population).12 In case of SAP, the criteria included chest pain for at least 6 months accompanied by evidence of severe CAD on CAG, with no clinically evident ischemic episodes during the week preceding arteriography.13 Exclusion criteria were a history of cardiomyopathy, valvular heart disease, severe renal failure (creatinine .2.5% mg), respiratory insufficiency, severe hepatic disease, malignant neoplasia or terminal cachexia, active infective diseases, peripheral angiopathy, cerebral vessel diseases and mental disorders or linguistic barriers that impeded adequate comprehension and collaboration. Data Collection Trained research assistants, who retrospectively reviewed all individual medical records, undertook the data collection. All data collection was conducted with quality control. Demographic characteristics (age and sex) and data regarding the presence of coronary risk factors (body mass index [BMI], hypertension, type 2 diabetes, hyperlipidemia, family history of CHD, smoking status and drinking history) were collected. In addition, laboratory measurements were conducted by the clinical laboratory of the First Affiliated Hospital of Zhengzhou University, including CK-MB, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP) and N-terminal probrain natriuretic peptide. The left-ventricular ejection fraction (%) was calculated by 2-dimensional echocardiography using a digital imaging system (Vivid-7; GE Medical System, Willoughby, OH). Measurements of CTRP-1 and IL-6 Blood samples used for the assessment of plasma CTRP-1 and IL-6 levels were obtained from all individuals by venipuncture after admission to the hospital and 15 minutes of bed rest and collected in tubes containing EDTA and aprotinin. Plasma samples were separated by centrifugation at 4°C at 3,000 rpm for 10 minutes, then subsequently frozen and stored at 280°C until assayed in a blinded manner in a single batch. The levels of expressed CTRP-1 were detected using enzyme-linked immunosorbent assay
kits (Elabscience Biotechnology Co Ltd, Wuhan, China), and IL-6 levels were measured by enzyme-linked immunosorbent assay (Nanjing Jiancheng Technology Industry Co Ltd, Nanjing, China). These assay kits had a lower limit of detection of 0.19 ng/L for CTRP-1 and 2.00 ng/L for IL-6. We performed every process of the experiment with strict quality control. And the quality control procedure for all blood samples and the detection of CTRP-1 and IL-6 were completed. More than 90% of our samples exhibited a normal odds ratio. Coronary Angiography CAG was performed using standard Judkins’ techniques. Two experienced interventional cardiologists who were unaware of the subjects’ clinical information performed the angiographic analysis. CHD was defined as the presence of a 50% or greater decrease in internal diameter in at least 1 main coronary artery. The Gensini’s score and the number of involved coronary branches were used to assess the extent and severity of CAD. According to the results of CAG, patients with CHD were further grouped according to the number of significantly stenotic vessels, defined as a decrease in internal diameter of more than 50%. These patients were grouped into single-vessel, double-vessel and triple-vessel disease groups. Statistical Analysis The data were analyzed with SPSS version 20.0 (IBM SPSS Inc, Chicago, IL). Continuous variables were presented as the mean 6 SD or median (interquartile range). The Shapiro-Wilk’s test was performed to evaluate normality. Categorical variables were presented as absolute and relative frequencies. Mean values in groups were compared using parametric statistics (Student’s t test and analysis of variance) or nonparametric statistics (MannWhitney and Kruskal-Wallis’ tests), depending on the distribution of the variable of interest. Fisher’s exact test was applied when comparing the number of patients with type 2 diabetes mellitus or hyperlipidemia in the ACS, SAP and control groups. Bonferroni’s correction was used to adjust for multiple comparisons. If distributions of both variables were normal, we selected the Pearson’s test for correlation analysis; and if the distributions of variables were not normal, the Spearman’s test was chosen. The univariate logistic regression analysis was first used to screen the significant independent variables and then followed by the multivariate logistic regression analysis with screened categorical variables and with or without CHD as dependent variables using the forward stepwise (conditional) method to identify the risk factors. P , 0.05 was considered statistically significant.
RESULTS Baseline Characteristics of Subjects Comparisons of the clinical and biochemical characteristics of the study subjects are shown in the Table 1. Among the 3 groups, there was no significant difference in gender, BMI, medical history of hypertension, family history of CAD, drinking history, heart rate, systolic blood pressure (SBP), TC, TG or LDL-C. A greater number of patients with ACS had a history of smoking compared with those in the SAP group (P , 0.01); in addition, subjects with ACS had significantly lower levels of HDL-C compared with the SAP and control groups (P , 0.01). Subjects with SAP were significantly older compared with the control group (P , 0.05), and the ACS group had a higher diastolic blood pressure (DBP) compared with the SAP group (P , 0.05). Much more patients had a history of type 2 diabetes mellitus and hyperlipidemia in the SAP group compared with the control group (P , 0.01), moreover the number of patients
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TABLE 1. Baseline characteristics of the study participants Clinical variables ACS group No. patients Male, n (%) Age, y BMI, kg/m2 Medical history Hypertension (%) Type 2 diabetes mellitus (%) Hyperlipidemia (%) Family history of CAD (%) Smoking history (%) Drinking history (%) Diagnostic tests SBP, mm Hg DBP, mm Hg Heart rate (/min) TC, mmol/L TG, mmol/L LDL-C, mmol/L HDL-C, mmol/L
41 28 (68.3) 55.63 6 9.54 23.1 (22.05–23.5) 7 7 4 6 18 15
(17.1) (17.1) (9.8)c (14.6) (43.9)c (36.6)
SAP group
Control group
40 23 (57.5) 57.35 6 10.53a 23.2 (22.63–23.5)
30 18 (60.0) 50.87 6 10.56 23.0 (21.68–23.5)
14 8 19 1 5 6
126.0 6 27.00 84.00 (80.00–88.00)d 78.02 6 6.80 4.39 (3.72–4.50) 1.81 (0.89–2.33) 2.50 (2.24–3.17) 0.85 (0.79–0.95)b,c
(35.0) (20.0)b (47.5)b (2.5) (12.5) (15.0)
132.70 6 17.63 80.00 (74.00–90.00) 73.48 6 11.68 4.35 (3.78–4.70) 1.40 (0.76–1.90) 2.62 (1.72–3.12) 1.09 (0.99–1.38)
10 (33.3) 0 0 4 (13.3) 8 (26.7) 7 (23.3) 136.53 6 19.85 80.00 (78.00–82.00) 75.83 6 9.18 4.23 (3.90–4.86) 1.36 (1.05–1.77) 2.62 (2.50–2.87) 1.16 (1.01–1.27)
P 0.581 0.030 0.481
x2 5 1.08 F 5 3.62 x2 5 1.47
0.147 ,0.01 ,0.01 0.144 0.007 0.079
x2 5 3.83 — — x2 5 3.88 x2 5 9.95 x2 5 5.08
0.122 0.023 0.100 0.697 0.266 0.654 ,0.01
F 5 2.14 x2 5 7.54 F 5 2.35 x2 5 0.72 x2 5 2.65 x2 5 0.85 x2 5 42.13
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.05 in comparison with control group. b P , 0.01 in comparison with control group. c P , 0.01 in comparison with SAP group. d P , 0.05 in comparison with SAP group. ACS, acute coronary syndrome; BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SAP, stable angina pectoris; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
with ACS had a history of hyperlipidemia was lower compared with those in the SAP group (P , 0.01) (Table 1). Comparison of Plasma Levels of CTRP-1 and IL-6 in Patients With CHD and the Control Group Comparisons of the plasma levels of CTRP-1 and IL-6, cardiac biochemical features and left ventricular function among patients with ACS or SAP and the healthy control group are shown in Table 2. The levels of CTRP-1 were
significantly increased in patients with ACS or SAP compared with control subjects (P , 0.01), and the expressed IL-6 was higher in the ACS or SAP group than the control group (P , 0.01). Interestingly, the concentrations of hs-CRP and N-terminal probrain natriuretic peptide in ACS were much higher than in the SAP and control groups (P , 0.01), as was the concentration of CK-MB in the ACS group (P , 0.05). The ACS group had a much lower level of left ventricular ejection fraction compared with the SAP and control groups (P , 0.01), whereas Gensini’s
TABLE 2. Comparisons of biochemical features, left ventricular function, plasma levels of CTRP-1 and IL-6 in patients with CHD and the control group ACS group SAP group Control group P No. patients CTRP-1, ng/L IL-6, ng/L hs-CRP, mg/dL NT-proBNP, pg/mL CKMB, U/L LVEF, % Gensini score
41 13.98 6 1.22a,b 223.14 (209.94, 52.84)a,b 6.41 (3.24–10.06)a,b 268.00 (213.00–425.00)a,b 15.00 (9.00–95.00)c,d 50.00 (44.00–60.00)a,b 61.00 (37.25–87.00)b
40 11.42 6 1.11b 116.20 (74.20–170.12)b 1.38 (0.92–2.54) 119.00 (68.25–188.50) 12.50 (5.25–21.00) 67.00 (63.25–68.00) 14.50 (3.87–32.62)
30 9.90 6 0.57 46.92 (36.38–57.61) 0.72 (0.29–2.37) 94.25 (69.00–143.50) 11.50 (6.00–20.00) 65.00 (50.00–68.50) 0
— ,0.01 ,0.01 ,0.01 ,0.01 0.048 ,0.01 ,0.01
F 5 140.56 x2 5 91.24 x2 5 42.29 x2 5 39.79 x2 5 6.06 x2 5 32.62 x2 5 25.08
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.01 in comparison with SAP group. b P , 0.01 in comparison with control group. c P , 0.05 in comparison with SAP group. d P , 0.05 in comparison with control group. ACS, acute coronary syndrome; SAP, stable angina pectoris; CTRP-1, C1q/TNF-related protein 1; IL-6, interleukin 6; hs-CRP, high-sensitivity C-reactive protein; LVEF, left ventricular ejection fraction.
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score, which could in part indicate the degree of coronary atherosclerosis, was higher in the ACS group than in the SAP group (P , 0.01). Comparisons of Plasma CTRP-1 and IL-6 in the Single-, Double- and Triple-Vessel Disease Groups Plasma levels of CTRP-1 were significantly increased in the single-vessel, double-vessel and triple-vessel disease groups compared with the control group (P , 0.01), and the singlevessel group had a much lower level of CTRP-1 compared with the double-vessel and triple-vessel disease groups (P , 0.01). However, there was no significant difference between the double-vessel and triple-vessel disease groups. The concentrations of IL-6 were much higher in the single-vessel, doublevessel and triple-vessel disease groups than in the control group (P , 0.01), and the expression of IL-6 in the single-vessel group was much lower than the double-vessel (P , 0.05) and the triple-vessel disease groups (P , 0.01). At the same time, a much higher expression of IL-6 was observed in the triplevessel disease group compared with the double-vessel group (P , 0.01) (Table 3). Correlations of CTRP-1 and IL-6 With the Cardiac Risk Factors in CHD The correlations of CTRP-1 and IL-6 with the cardiac risk factors in CHD are listed in Table 4. The level of CTRP-1 in patients was positively correlated with the level of IL-6 (r 5 0.667, P , 0.01). Furthermore, both the concentrations of CTRP-1 and IL-6 were negatively correlated with the HDL-C (r 5 20.432, P , 0.01; r 5 20.514, P , 0.01), whereas the levels of CTRP-1 and IL-6 were positively correlated with the hs-CRP (r 5 0.520, P , 0.01; r 5 0.546, P , 0.01). The expression of IL-6 was positively correlated with the heart rate (r 5 0.241, P 5 0.03). However, the level of CTRP-1 had no obvious correlation with the heart rate, and neither the expression of CTRP-1 nor that of IL-6 was significantly correlated with the levels of BMI, SBP, DBP, TC, TG or LDL-C. Logistic Regression Analysis for the Detection of an Independent Predictor of CHD To determine the association of possible risk factors for CHD and the occurrence of CHD, we first performed a univariate logistic regression analysis to screen the significant independent variables followed by a multivariate logistic regression analysis with age, gender, smoking history,
hs-CRP, HDL-C, IL-6 and CTRP-1 as covariates and with or without CHD as dependent variables. With forward stepwise (conditional) method: Wald, the binary logistic regression analysis, showed that increases in CTRP-1 and IL-6 were powerful predictive factors for the occurrence of CHD (Table 5).
DISCUSSION Inflammation is a primary factor promoting the initiation of coronary plaques, their unstable progression and their final disruption,14 which together underlie the pathogenesis of ACS. Immune cells dominate early atherosclerotic lesions. Their effector molecules accelerate the progression of the lesions, and the activation of inflammation can elicit ACS, which is an acute stage of CHD, AMI and UAP.1 Patients with a more obvious vascular inflammatory response have a poorer outcome. It is now well established that adipose tissue is not only involved in energy storage but also functions as an endocrine organ that secretes various bioactive substances. Factors that are secreted by adipose tissue are collectively referred to as adipokines15,16 and function in a pro- or anti-inflammation capacity in metabolic dysfunction. For instance, the TNF produced by monocytes and macrophages was identified as a proinflammatory product of adipose tissue17 that promotes the progression of diabetes, obesity and atherosclerosis. Recently, a new protein family of secreted adipokines, C1q/TNF-related protein (CTRP), was discovered. This family has been reported to provide a link between inflammation and metabolism.4 Each CTRP family member has its own unique tissue expression profile and function in regulating glucose and/or fat metabolism.18 As a family member of CTRP, fat tissue CTRP-1 expression was in vivo stimulated by rosiglitazone, a drug with anti-inflammatory properties and a high-affinity ligand for the adipogenic transcription factor peroxisome proliferator-activated receptor.5 In addition, IL-1b strongly upregulates CTRP-1 in the epididymal fat of SD rats, an effect that is, enhanced by TNF-a,7 both of which are induced by LPS. These data indicate that adipose tissue CTRP-1 expression is induced in inflammation. Whereas SVC is a major source of CTRP-1, and LPS-mediated upregulation in fat might also be related to enhanced synthesis in macrophages, whether higher CTRP-1 expression is associated with increased adipocyte and/or SVC synthesis or simply reflects altered SVC composition in obesity has not yet been determined. These findings suggest that CTRP-1
TABLE 3. Comparisons of plasma CTRP-1 and IL-6 in single, double and triple-vessel diseases n CTRP-1, ng/L 11.58 6 1.21a,b,c 12.93 6 1.62a 13.38 6 1.72a 9.90 6 0.57 ,0.01 F 5 40.60
IL-6, ng/L 119.02 159.99 209.94 46.92
(73.89–178.61)a,c,d (80.77–249.39)a,c (202.25–239.01)a (36.38–57.61) ,0.01 x2 5 72.50
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Singe-vessel lesion group, n (%) Double-vessel lesion group, n (%) Triple-vessel lesion group, n (%) Control group P
26 16 29 30
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.01 in comparison with control group. b P , 0.01 in comparison with double-vessel group. c P , 0.01 in comparison with triple-vessel group. d P , 0.05 in comparison with double-vessel group. CTRP-1, C1q/TNF-related protein-1; IL-6, interleukin 6.
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TABLE 4. Correlation analysis of circulating CTRP-1and IL-6 levels with cardiac risk factors CTRP1 IL-6
2
BMI, kg/m SBP, mm Hg DBP, mm Hg Heart rate/min TC, mmol/L TG, mmol/L LDL-C, mmol/L HDL-C, mmol/L hs-CRP, mg/dL CTRP-1 IL-6
r
P
r
P
20.043 20.169 0.122 0.207 0.018 0.148 0.105 20.432 0.520 — 0.667
0.703 0.131 0.276 0.063 0.873 0.188 0.353 ,0.01 ,0.01 — ,0.01
20.094 20.075 0.073 0.241 20.154 0.111 0.007 20.514 0.546 0.667 —
0.403 0.506 0.517 0.03 0.171 0.324 0.947 ,0.01 ,0.01 ,0.01 —
BMI, body mass index; CTRP-1, C1q/TNF-related protein-1; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
is increased in obesity and that its upregulation might be related to the inflammatory aspect of this condition.3 In our study, CTRP-1 expression was much higher in the ACS group compared with the SAP or control groups, which suggests that CTRP-1 may function as an inflammatory agent in the acute phase of CHD and, as such, impact the stability of atheromatous plaque. Recently, Peterson et al19 demonstrated that CTRP-1 could act through the activation of AMP-activated protein kinase (AMPK) and inhibition of acetyl-CoA carboxylase in muscle to enhance fatty acid oxidation. Furthermore, AMPK has been noted to mediate preconditioning in isolated cardiomyocytes under hypoxic conditions.20 Increased AMPK activity likely compensates for the ischemic condition by upregulating GLUT4 translocation and enhancing fatty acid oxidation.21,22 Furthermore, Chalupova et al reported that circulating CTRP-1 levels were elevated in patients with metabolic syndrome compared with healthy people. In addition, CTRP-1 is correlated with glucose levels, HbA1c and BMI in metabolic syndrome patients,23 indicating that CTRP-1 is intimately linked to cardiovascular risk factors. In our study, we explored the correlations between the levels of CTRP-1 and cardiacrelated factors, and we found that CTRP-1 was negatively correlated with the cardiac protective factor HDL-C, which may provide additional evidence that CTRP-1 is closely related to cardiac risk factors. Simultaneously, the CTRP-1 concentrations were much higher in the groups with coronary vessel lesions (single, double or triple) compared with the healthy control group. And there was a significant difference between the single-vessel lesion group and double-vessel or triple-vessel groups, which may reflect a degree of coronary vessel stenosis.
Moreover, Lasser et al8 demonstrated that CTRP-1 could specifically bind to fibrillar collagen type I and block collageninduced platelet activation and aggregation. These observations indicate that CTRP-1 may play a role in the process of thrombosis, a key contributor to the development and aggravation of CHD. In this study, IL-6 cytokines and their signaling events were shown to contribute to both atherosclerotic plaque development and plaque destabilization through a variety of mechanisms.24 IL-6 is the eponym of an entire cytokine family, including molecules such as IL-11, IL-27, leukemia inhibitory factor, oncostatin M and several others.25 The expression of IL-6, a major determinant of the production of acute-phase proteins, is increased in a number of clinical conditions, such as tissue injury, infections, malignant neoplasms, ischemic diseases and trauma.26 One clinical trial, a case-control study of 294 patients with clinical ACS (group I) and clinically SAP (group II), showed the median IL-6 levels to be significantly higher in group I than in group II (P , 0.05).27 Experimentally, treatment with recombinant IL-6 was found to exacerbate the degree of atherosclerosis in atherosclerosis-prone ApoE-deficient mice and was accompanied by increased levels of other proinflammatory cytokines.28 Furthermore, it has been reported that trans-IL-6 receptor binding may alter intracellular signaling. Blocking of IL-6 receptor binding may be pathogenic in AMI, as IL-6 and the soluble IL-6 receptor were elevated, whereas IL-6R mRNA was decreased, in patients with AMI when compared with patients with stable CAD and controls.29 Using multivariable-adjusted Cox proportional hazards regression analyses in a prospective cohort study of 718 patients with CAD, the authors found that serum IL-6 was significantly associated with all-cause cardiovascular mortality.30 In this study, we demonstrated that IL-6 concentrations were significantly increased in patients with ACS or SAP compared with the healthy subjects. The IL-6 concentration in the triplevessel lesions was significantly higher compared with single- or double-vessel lesions, as well as a higher expression in the double-vessel lesion group compared with the single-vessel lesion group. This result indicated that IL-6 acted as an inflammatory factor and may be useful in evaluating the severity of CHD from the perspective of both the evolution of CHD and the degree of coronary artery stenosis. In addition, we conducted a correlation analysis on the levels of CTRP-1 and IL-6 with all cardiac-related factors that were evaluated in our study, including BMI, SBP, DBP, heart rate, TC, TG, LDL-C, HDL-C and hs-CRP. We found that the concentrations of both CTRP-1 and IL-6 were negatively correlated with HDL-C, whereas the levels of CTRP-1 and IL-6 were positively correlated with hs-CRP. We also detected a positive correlation between concentrations of CTRP-1 and IL-6 in patients with CHD. Although the detailed mechanism of how the cytokines interact is still unclear, there are 2 possible explanations for these correlations. First, atherosclerosis is a complex chronic inflammatory and metabolic disease that is, due to the interaction of several cellular components of the immune system involving a multitude of immune cell subsets
TABLE 5. Logistic regression analysis for the detection of independent relationship with the occurrence of CHD Variables b SE Wald x2 OR 95% CI Constant CTRP-1 IL-6
223.98 1.88 0.06
6.86 0.63 0.20
12.23 8.95 9.49
0.0 6.57 1.06
— 1.91–22.54 1.02–1.11
P ,0.01 0.003 0.002
CI, confidence interval; CHD, coronary heart disease; CTRP-1, C1q/TNF-related protein-1; IL-6, interleukin 6; OR, odds ratio; SE, standard error.
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together with soluble molecules. This complex process leads to thickening of the arterial wall.31 CTRP-1 is secreted by stromal vascular SVC composed of adipose-tissue macrophages, and its expression is stimulated by TNF-a and IL-1b, which are both the inflammatory mediators that contribute to the process of CHD.7,32,33 In human coronary atherosclerotic plaques, IL-6 seems to stimulate macrophages and regulate the expression of adhesion molecules and other cytokines such as IL-1b and TNF-a, potentially enhancing the inflammatory reaction.10 Therefore, IL-6 may enhance the expression of CTRP-1, further adding to the inflammation associated with atherosclerotic plaques. Both CTRP-1 and IL-6 were positively correlated with the inflammatory factor hs-CRP, further supporting the role of CTRP-1 and IL-6 as inflammatory mediators. Second, CTRP1 is an adipocytokine associated with metabolic syndrome and obesity, which have similar risk factors as cardiovascular disease.23 IL-6 is also produced in the adipose tissue, linking obesity to a state of chronic low-level inflammation as a potential trigger for cardiovascular and metabolic diseases.24 In this study, both CTRP-1 and IL-6 were negatively correlated with the protective factor HDL-C, suggesting that CTRP-1 and IL-6 may both play an important role in cardiac and metabolic risk factors that influence the development of atherosclerosis. Finally, these 2 factors interact and thereby amplify the development of atherosclerosis, which might explain why both CTRP-1 and IL-6 are more highly expressed in the plasma of patients compared with the healthy group. The progression of CHD is the result of unstable coronary atherosclerotic plaques and the stenosis of coronary arterial atherosclerosis, both of which are closely linked with unbalanced inflammatory cytokines. We have described the plasma levels of CTRP-1 and IL-6 in CHD of different levels of severity. Dysregulated secretion of CTRP-1 and IL-6 is speculated to be involved in the pathogenesis of CHD, and the variation in CTRP-1 and IL-6 expression in CHD suggests that these proteins might be useful biomarkers of disease severity. In addition, in the logistic analysis for the detection of independent factors associated with the occurrence of CHD, we found that CTRP-1 and IL-6 may also play roles in predicting the development of CHD. Some limitations of the study must be considered. First, the study was conducted in a single center, and the number of subjects was small. Furthermore, plasma levels of CTRP-1 and IL-6 were not measured at different time points; thus, changes over time or potential fluctuations due to various medications were not examined. In addition, mechanisms through which CTRP-1 could influence the stability of coronary atherosclerotic plaques and the process of coronary arterial atherosclerosis as well as possible ischemia/reperfusion through the AMPK pathway are worthy of exploration. Hence, larger clinical trials, longer follow-up and research to uncover underlying mechanisms are needed to validate the findings of this study. In conclusion, patients with CHD (ACS or SAP) have significantly increased circulating concentrations of CTRP-1 and IL-6. This study suggests that the variation in plasma CTRP-1 and IL-6 concentrations may play an important role in reflecting the degree of inflammation in CHD and the severity of coronary arterial atherosclerosis. The combined determination of CTRP-1 and IL-6 may play a role in predicting the development of CHD. REFERENCES 1. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95.
2. Falcão-Pires I, Castro-Chaves P, Miranda-Silva D, et al. Physiological, pathological and potential therapeutic roles of adipokines. Drug Discov Today 2012;17:880–9. 3. Schäffler A, Buechler C. CTRP family: linking immunity to metabolism. Trends Endocrinol Metab 2012;23:194–204. 4. Wong GW, Wang J, Hug C, et al. A family of Acrp30/adiponectin structural and functional paralogs. Proc Natl Acad Sci U S A 2004;101: 10302–7. 5. Wong GW, Krawczyk SA, Kitidis-Mitrokostas C, et al. Molecular, biochemical and functional characterizations of C1q/TNF family members: adipose-tissue-selective expression patterns, regulation by PPARgamma agonist, cysteine-mediated oligomerizations, combinatorial associations and metabolic functions. Biochem J 2008;416:161–77. 6. Innamorati G, Whang MI, Molteni R, et al. GIP, a G-protein-coupled receptor interacting protein. Regul Pept 2002;109:173–9. 7. Kim KY, Kim HY, Kim JH, et al. Tumor necrosis factor-alpha and interleukin-1beta increases CTRP1 expression in adipose tissue. FEBS Lett 2006;580:3953–60. 8. Lasser G, Guchhait P, Ellsworth JL, et al. C1qTNF-related protein-1 (CTRP-1): a vascular wall protein that inhibits collagen-induced platelet aggregation by blocking VWF binding to collagen. Blood 2006;107:423–30. 9. Shu J, Ren N, Du JB, et al. Increased levels of interleukin-6 and matrix metalloproteinase-9 are of cardiac origin in acute coronary syndrome. Scand Cardiovasc J 2007;41:149–54. 10. Schieffer B, Schieffer E, Hilfiker-Kleiner D, et al. Expression of angiotension II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation 2000;101:1372–8. 11. Thygesen K, Alpert JS, Jaffe AS, et al. Third universal definition of myocardial infarction. Circulation 2012;126:2020–35. 12. Anderson JL, Adams CD, Antman EM, et al. ACC/AHA 2007 guidelines for the management of patients with unstable angina/nonST-Elevation myocardial infarction. J Am Coll Cardiol 2007;50:1–157. 13. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACPASIM guidelines for the management of patients with chronic stable angina: executive summary and recommendations. Circulation 1999;99:2829–48. 14. Zakynthinos E, Pappa N. Inflammatory biomarkers in coronary artery disease. J Cardiol 2009;53:317–333. 15. Ouchi N, Kihara S, Funahashi T, et al. Obesity, adiponectin and vascular inflammatory disease. Curr Opin Lipidol 2003;14:561–6. 16. Berg AH, Scherer PE. Adipose tissue, inflammation, and cardiovascular disease. Circ Res 2005;96:939–49. 17. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance. Science 1993;259:87–91. 18. Seldin MM, Tan SY, Wong GW. Metabolic function of the CTRP family of hormones. Rev Endocr Metab Disord 2014;15:111–123. 19. Peterson JM, Aja S, Wei Z, et al. CTRP1 protein enhances fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and acetyl-CoA carboxylase (ACC) inhibition. J Biol Chem 2012;287: 1576–87. 20. Sukhodub A, Jovanovic S, Du Q, et al. AMP-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K(+) channels. J Cell Physiol 2007;210:224–36. 21. Kudo N, Barr AJ, Barr RL, et al. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 59-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 1995;270: 17513–20.
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22. Russell RR III, Bergeron R, Shulman GI, et al. Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 1999;277(2 pt 2):H643–9. 23. Chalupova L, Zakovska A, Adamcova K. Development of a novel enzyme-linked immunosorbent assay (ELISA) for measurement of serum CTRP1 A pilot study: measurement of serum CTRP1 in healthy donors and patients with metabolic syndrome. Clin Biochem 2013;46: 73–8.
interleukin-6 in patients with acute coronary syndrome and stable angina pectoris. Coron Artery Dis 2008;19:15–9. 28. Huber SA, Sakkinen P, Conze D, et al. Interleukin-6 exacerbates early atherosclerosis in mice. Arterioscler Thromb Vasc Biol 1999;19:2364–7. 29. Anderson DR, Poterucha JT, Mikuls TR, et al. IL-6 and its receptors in coronary artery disease and acute myocardial infarction. Cytokine 2013;62:395–400.
24. Schuett H, Luchtefeld M, Grothusen C, et al. How much is too much? Interleukin-6 and its signalling in atherosclerosis. Thromb Haemost 2009;102:215–22.
30. Su D, Li Z, Li X, et al. Association between serum interleukin-6 concentration and mortality in patients with coronary artery disease. Mediators Inflamm 2013;2013:726178.
25. Taga T, Kishimoto T. Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol 1997;15:797–819.
31. Garrido-Urbani S, Meguenani M, Montecucco F, et al. Immunological aspects of atherosclerosis. Semin Immunopathol 2014;36:73–91.
26. Papanicolaou DA, Wilder RL, Manolagas SC, et al. The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 1998; 128:127–37.
32. Fragoso Lona JM, Sierra Martínez M, Vargas Alarcón G, et al. Tumor necrosis factor alfa in cardiovascular diseases: molecular biology and genetics [in Spanish]. Gac Med Mex 2013;149:521–30.
27. Ozdemir O, Gundogdu F, Karakelleoglu S, et al. Comparison of serum levels of inflammatory markers and allelic variant of
33. Qamar A, Rader DJ. Effect of interleukin 1b inhibition in cardiovascular disease. Curr Opin Lipidol 2012;23:548–53.
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Plasma Levels of C1q/TNF-Related Protein 1 and Interleukin 6 in Patients With Acute Coronary Syndrome or Stable Angina Pectoris Jun-Nan Tang, PhD, De-Liang Shen, PhD, Cong-Lin Liu, PhD, Xiao-Fang Wang, PhD, Li Zhang, MD, Xue-Xi Xuan, MD, Ling-Ling Cui, PhD and Jin-Ying Zhang, PhD
Abstract: Background: C1q/TNF-related protein 1 (CTRP-1), a novel adipocyte factor, may participate in the mechanisms of metabolism and inflammation. Interleukin 6 (IL-6) is a proinflammatory cytokine that is correlated with the severity of coronary heart disease (CHD). In this study, we focused on the levels of CTRP-1 and IL-6 in patients with CHD. Methods: Circulating CTRP-1 and IL-6 levels were measured using enzyme-linked immunosorbent assay in 81 patients with acute coronary syndrome (n 5 41) or stable angina pectoris (n 5 40). CTRP1 and IL-6 levels were also examined in 30 healthy individuals (control group). We examined the correlations between the levels of CTRP-1 and IL-6 and cardiac risk factors in CHD. Logistic regression analysis was performed to screen for factors that predict CHD. Results: Both CTRP-1 and IL-6 concentrations were increased in the acute coronary syndrome or stable angina pectoris group compared with the control group (P , 0.01). Both plasma levels of CTRP-1 and IL-6 in the single-, double- and triple-vessel lesion group were higher compared with the control group (P , 0.01). CTRP-1 levels were positively correlated with IL-6 (r 5 0.667, P , 0.01) and high-sensitivity C-reactive protein levels (r 5 0.520, P , 0.01) and negatively correlated with HDL-C (r 5 20.432, P , 0.01). The logistic regression analysis showed that increases in CTRP-1 and IL-6 levels may be powerful predictors of CHD. Conclusions: The variation of plasma CTRP-1 and IL-6 concentrations may play an important role in reflecting the degree of inflammation in CHD and the severity of coronary arterial atherosclerosis. This potential suggests that evaluating CTRP-1 and IL-6 in combination may aid in predicting the occurrence of CHD. Key Indexing Terms: C1q/TNF-related protein 1; Interleukin 6; Coronary heart disease; Acute coronary syndrome; Stable angina pectoris. [Am J Med Sci 2015;349(2):130–136.]
C
oronary heart disease (CHD) is one of the major cardiovascular diseases, which has seriously impacted human health globally. CHD is an inflammatory disease in which immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial tree.1 As a vital endocrine organ, adipose tissue secretes various types of bioactive substances, known as adipokines, that play important roles in the pathogenesis of obesity, From the Department of Cardiology (J-NT, D-LS, C-LL, X-FW, LZ, X-XX, J-YZ), the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; Institute of Clinical Medicine (J-NT), the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; and College of Public Health (L-LC), Zhengzhou University, Zhengzhou, China. Submitted May 20, 2014; accepted in revised form September 25, 2014. Supported by the Science and Technology Talents Team Construction Program of Zhengzhou City-Science and Technology Talents (131PLJRC670) and the Science and Technology Research Projects of Henan Province (13A320623). The authors have no conflicts of interest to disclose. Correspondence: Jin-Ying Zhang, PhD, Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China (E-mail: [email protected]).
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diabetes, hypertension and cardiovascular diseases.2 Recently, a new protein family secreted by adipokines, C1q/TNF-related protein (CTRP), was cloned on the basis of its sequence homology with adiponectin.3 This family of adiponectin paralogs may be involved in energy homeostasis and obesity-related inflammation.4 C1q/TNF-related protein 1 (CTRP-1), a member of the CTRP family, is secreted by stromal vascular cells (SVCs) composed of adipose-tissue macrophages, preadipocytes and endothelial cells.5 In human tissues, CTRP-1 mRNA is expressed more highly in the heart than in many other tissues such as the liver and kidney.6 Increased CTRP-1 gene expression can be indirectly induced by lipopolysaccharide (LPS) in epididymal adipose tissue and is regulated by the inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) in association with a decrease in adiponectin mRNA expression,7 indicating that adipose tissue CTRP-1 expression is induced in inflammation. In addition, Lasser et al8 demonstrated that CTRP-1 could specifically bind to fibrillar collagen type I and block collagen-induced platelet activation and aggregation. These observations indicate that CTRP-1 may play a role in the process of plaque formation in CHD. Interleukin 6 (IL-6) is a proinflammatory cytokine that is triggered by vulnerable plaque or necrotic myocardium and is correlated with the severity of coronary artery disease (CAD).9 In patients with CAD, elevated circulating IL-6 concentrations may be involved in the function of macrophage/foam cells present in atheromatous plaques.10 At present, data are limited regarding the correlation between plasma concentrations of CTRP-1 and IL-6 and the severity of CHD. Therefore, in this study, we measured plasma concentrations of these biomarkers and investigated their relationship with disease severity in patients with CHD.
METHODS Study Participants and Definition of Coronary Heart Disease The study was approved by the Institution Review Board of the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from every participant. From May 1, 2013, to February 1, 2014, 81 consecutive patients with CHD (51 men; mean age: 56.48 6 10.02 years; range, 39–85) were recruited from the Department of Cardiology in the First Affiliated Hospital of Zhengzhou University. All patients had CHD confirmed by coronary angiography (CAG). The enrolled patients were classified into 2 groups: a group with acute coronary syndrome (ACS) and a group with stable angina pectoris (SAP). The ACS group consisted of 20 patients with ST-elevation acute myocardial infarction and 21 patients with unstable angina pectoris (UAP). The SAP group consisted of 40 patients with SAP. Percutaneous coronary
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intervention was performed in 81 patients with ACS and SAP. During the same period, 30 healthy subjects (18 men; mean age: 50.87 6 10.56 years; range, 34–77) were recruited as a control group, excluding individuals with a history of CVD (myocardial infarction, unstable angina, stroke or cardiovascular revascularization), type 2 diabetes, stage 2 hypertension (resting blood pressure $160/100 mm Hg), malignancy or severe renal or hepatic disease. Acute myocardial infarction (AMI) was defined as a typical increase and gradual decrease of biochemical markers of myocardial necrosis (detection of a rise and/or fall of cardiac biomarkers such as CK-MB and/or troponin-T with at least 1 value above the 99th percentile upper reference limit) and at least 1 of the following: Ischemic symptoms, electrocardiogram changes indicative of new ischemia (new ST-T changes or new left bundle branch block), development of pathologic Q waves on electrocardiogram and imaging evidence of a new loss of viable myocardium or new regional wall motion abnormality.11 The diagnostic criteria for UAP included chest pain at rest with either a ST segment depression of at least 0.1 mV or a T-wave inversion in 2 or more continuous electrocardiographic leads and no biomarkers of myocardial necrosis (based on 2 or more blood samples collected at least 6 hours apart, with a reference limit of the 99th percentile of the normal population).12 In case of SAP, the criteria included chest pain for at least 6 months accompanied by evidence of severe CAD on CAG, with no clinically evident ischemic episodes during the week preceding arteriography.13 Exclusion criteria were a history of cardiomyopathy, valvular heart disease, severe renal failure (creatinine .2.5% mg), respiratory insufficiency, severe hepatic disease, malignant neoplasia or terminal cachexia, active infective diseases, peripheral angiopathy, cerebral vessel diseases and mental disorders or linguistic barriers that impeded adequate comprehension and collaboration. Data Collection Trained research assistants, who retrospectively reviewed all individual medical records, undertook the data collection. All data collection was conducted with quality control. Demographic characteristics (age and sex) and data regarding the presence of coronary risk factors (body mass index [BMI], hypertension, type 2 diabetes, hyperlipidemia, family history of CHD, smoking status and drinking history) were collected. In addition, laboratory measurements were conducted by the clinical laboratory of the First Affiliated Hospital of Zhengzhou University, including CK-MB, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP) and N-terminal probrain natriuretic peptide. The left-ventricular ejection fraction (%) was calculated by 2-dimensional echocardiography using a digital imaging system (Vivid-7; GE Medical System, Willoughby, OH). Measurements of CTRP-1 and IL-6 Blood samples used for the assessment of plasma CTRP-1 and IL-6 levels were obtained from all individuals by venipuncture after admission to the hospital and 15 minutes of bed rest and collected in tubes containing EDTA and aprotinin. Plasma samples were separated by centrifugation at 4°C at 3,000 rpm for 10 minutes, then subsequently frozen and stored at 280°C until assayed in a blinded manner in a single batch. The levels of expressed CTRP-1 were detected using enzyme-linked immunosorbent assay
kits (Elabscience Biotechnology Co Ltd, Wuhan, China), and IL-6 levels were measured by enzyme-linked immunosorbent assay (Nanjing Jiancheng Technology Industry Co Ltd, Nanjing, China). These assay kits had a lower limit of detection of 0.19 ng/L for CTRP-1 and 2.00 ng/L for IL-6. We performed every process of the experiment with strict quality control. And the quality control procedure for all blood samples and the detection of CTRP-1 and IL-6 were completed. More than 90% of our samples exhibited a normal odds ratio. Coronary Angiography CAG was performed using standard Judkins’ techniques. Two experienced interventional cardiologists who were unaware of the subjects’ clinical information performed the angiographic analysis. CHD was defined as the presence of a 50% or greater decrease in internal diameter in at least 1 main coronary artery. The Gensini’s score and the number of involved coronary branches were used to assess the extent and severity of CAD. According to the results of CAG, patients with CHD were further grouped according to the number of significantly stenotic vessels, defined as a decrease in internal diameter of more than 50%. These patients were grouped into single-vessel, double-vessel and triple-vessel disease groups. Statistical Analysis The data were analyzed with SPSS version 20.0 (IBM SPSS Inc, Chicago, IL). Continuous variables were presented as the mean 6 SD or median (interquartile range). The Shapiro-Wilk’s test was performed to evaluate normality. Categorical variables were presented as absolute and relative frequencies. Mean values in groups were compared using parametric statistics (Student’s t test and analysis of variance) or nonparametric statistics (MannWhitney and Kruskal-Wallis’ tests), depending on the distribution of the variable of interest. Fisher’s exact test was applied when comparing the number of patients with type 2 diabetes mellitus or hyperlipidemia in the ACS, SAP and control groups. Bonferroni’s correction was used to adjust for multiple comparisons. If distributions of both variables were normal, we selected the Pearson’s test for correlation analysis; and if the distributions of variables were not normal, the Spearman’s test was chosen. The univariate logistic regression analysis was first used to screen the significant independent variables and then followed by the multivariate logistic regression analysis with screened categorical variables and with or without CHD as dependent variables using the forward stepwise (conditional) method to identify the risk factors. P , 0.05 was considered statistically significant.
RESULTS Baseline Characteristics of Subjects Comparisons of the clinical and biochemical characteristics of the study subjects are shown in the Table 1. Among the 3 groups, there was no significant difference in gender, BMI, medical history of hypertension, family history of CAD, drinking history, heart rate, systolic blood pressure (SBP), TC, TG or LDL-C. A greater number of patients with ACS had a history of smoking compared with those in the SAP group (P , 0.01); in addition, subjects with ACS had significantly lower levels of HDL-C compared with the SAP and control groups (P , 0.01). Subjects with SAP were significantly older compared with the control group (P , 0.05), and the ACS group had a higher diastolic blood pressure (DBP) compared with the SAP group (P , 0.05). Much more patients had a history of type 2 diabetes mellitus and hyperlipidemia in the SAP group compared with the control group (P , 0.01), moreover the number of patients
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TABLE 1. Baseline characteristics of the study participants Clinical variables ACS group No. patients Male, n (%) Age, y BMI, kg/m2 Medical history Hypertension (%) Type 2 diabetes mellitus (%) Hyperlipidemia (%) Family history of CAD (%) Smoking history (%) Drinking history (%) Diagnostic tests SBP, mm Hg DBP, mm Hg Heart rate (/min) TC, mmol/L TG, mmol/L LDL-C, mmol/L HDL-C, mmol/L
41 28 (68.3) 55.63 6 9.54 23.1 (22.05–23.5) 7 7 4 6 18 15
(17.1) (17.1) (9.8)c (14.6) (43.9)c (36.6)
SAP group
Control group
40 23 (57.5) 57.35 6 10.53a 23.2 (22.63–23.5)
30 18 (60.0) 50.87 6 10.56 23.0 (21.68–23.5)
14 8 19 1 5 6
126.0 6 27.00 84.00 (80.00–88.00)d 78.02 6 6.80 4.39 (3.72–4.50) 1.81 (0.89–2.33) 2.50 (2.24–3.17) 0.85 (0.79–0.95)b,c
(35.0) (20.0)b (47.5)b (2.5) (12.5) (15.0)
132.70 6 17.63 80.00 (74.00–90.00) 73.48 6 11.68 4.35 (3.78–4.70) 1.40 (0.76–1.90) 2.62 (1.72–3.12) 1.09 (0.99–1.38)
10 (33.3) 0 0 4 (13.3) 8 (26.7) 7 (23.3) 136.53 6 19.85 80.00 (78.00–82.00) 75.83 6 9.18 4.23 (3.90–4.86) 1.36 (1.05–1.77) 2.62 (2.50–2.87) 1.16 (1.01–1.27)
P 0.581 0.030 0.481
x2 5 1.08 F 5 3.62 x2 5 1.47
0.147 ,0.01 ,0.01 0.144 0.007 0.079
x2 5 3.83 — — x2 5 3.88 x2 5 9.95 x2 5 5.08
0.122 0.023 0.100 0.697 0.266 0.654 ,0.01
F 5 2.14 x2 5 7.54 F 5 2.35 x2 5 0.72 x2 5 2.65 x2 5 0.85 x2 5 42.13
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.05 in comparison with control group. b P , 0.01 in comparison with control group. c P , 0.01 in comparison with SAP group. d P , 0.05 in comparison with SAP group. ACS, acute coronary syndrome; BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SAP, stable angina pectoris; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
with ACS had a history of hyperlipidemia was lower compared with those in the SAP group (P , 0.01) (Table 1). Comparison of Plasma Levels of CTRP-1 and IL-6 in Patients With CHD and the Control Group Comparisons of the plasma levels of CTRP-1 and IL-6, cardiac biochemical features and left ventricular function among patients with ACS or SAP and the healthy control group are shown in Table 2. The levels of CTRP-1 were
significantly increased in patients with ACS or SAP compared with control subjects (P , 0.01), and the expressed IL-6 was higher in the ACS or SAP group than the control group (P , 0.01). Interestingly, the concentrations of hs-CRP and N-terminal probrain natriuretic peptide in ACS were much higher than in the SAP and control groups (P , 0.01), as was the concentration of CK-MB in the ACS group (P , 0.05). The ACS group had a much lower level of left ventricular ejection fraction compared with the SAP and control groups (P , 0.01), whereas Gensini’s
TABLE 2. Comparisons of biochemical features, left ventricular function, plasma levels of CTRP-1 and IL-6 in patients with CHD and the control group ACS group SAP group Control group P No. patients CTRP-1, ng/L IL-6, ng/L hs-CRP, mg/dL NT-proBNP, pg/mL CKMB, U/L LVEF, % Gensini score
41 13.98 6 1.22a,b 223.14 (209.94, 52.84)a,b 6.41 (3.24–10.06)a,b 268.00 (213.00–425.00)a,b 15.00 (9.00–95.00)c,d 50.00 (44.00–60.00)a,b 61.00 (37.25–87.00)b
40 11.42 6 1.11b 116.20 (74.20–170.12)b 1.38 (0.92–2.54) 119.00 (68.25–188.50) 12.50 (5.25–21.00) 67.00 (63.25–68.00) 14.50 (3.87–32.62)
30 9.90 6 0.57 46.92 (36.38–57.61) 0.72 (0.29–2.37) 94.25 (69.00–143.50) 11.50 (6.00–20.00) 65.00 (50.00–68.50) 0
— ,0.01 ,0.01 ,0.01 ,0.01 0.048 ,0.01 ,0.01
F 5 140.56 x2 5 91.24 x2 5 42.29 x2 5 39.79 x2 5 6.06 x2 5 32.62 x2 5 25.08
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.01 in comparison with SAP group. b P , 0.01 in comparison with control group. c P , 0.05 in comparison with SAP group. d P , 0.05 in comparison with control group. ACS, acute coronary syndrome; SAP, stable angina pectoris; CTRP-1, C1q/TNF-related protein 1; IL-6, interleukin 6; hs-CRP, high-sensitivity C-reactive protein; LVEF, left ventricular ejection fraction.
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score, which could in part indicate the degree of coronary atherosclerosis, was higher in the ACS group than in the SAP group (P , 0.01). Comparisons of Plasma CTRP-1 and IL-6 in the Single-, Double- and Triple-Vessel Disease Groups Plasma levels of CTRP-1 were significantly increased in the single-vessel, double-vessel and triple-vessel disease groups compared with the control group (P , 0.01), and the singlevessel group had a much lower level of CTRP-1 compared with the double-vessel and triple-vessel disease groups (P , 0.01). However, there was no significant difference between the double-vessel and triple-vessel disease groups. The concentrations of IL-6 were much higher in the single-vessel, doublevessel and triple-vessel disease groups than in the control group (P , 0.01), and the expression of IL-6 in the single-vessel group was much lower than the double-vessel (P , 0.05) and the triple-vessel disease groups (P , 0.01). At the same time, a much higher expression of IL-6 was observed in the triplevessel disease group compared with the double-vessel group (P , 0.01) (Table 3). Correlations of CTRP-1 and IL-6 With the Cardiac Risk Factors in CHD The correlations of CTRP-1 and IL-6 with the cardiac risk factors in CHD are listed in Table 4. The level of CTRP-1 in patients was positively correlated with the level of IL-6 (r 5 0.667, P , 0.01). Furthermore, both the concentrations of CTRP-1 and IL-6 were negatively correlated with the HDL-C (r 5 20.432, P , 0.01; r 5 20.514, P , 0.01), whereas the levels of CTRP-1 and IL-6 were positively correlated with the hs-CRP (r 5 0.520, P , 0.01; r 5 0.546, P , 0.01). The expression of IL-6 was positively correlated with the heart rate (r 5 0.241, P 5 0.03). However, the level of CTRP-1 had no obvious correlation with the heart rate, and neither the expression of CTRP-1 nor that of IL-6 was significantly correlated with the levels of BMI, SBP, DBP, TC, TG or LDL-C. Logistic Regression Analysis for the Detection of an Independent Predictor of CHD To determine the association of possible risk factors for CHD and the occurrence of CHD, we first performed a univariate logistic regression analysis to screen the significant independent variables followed by a multivariate logistic regression analysis with age, gender, smoking history,
hs-CRP, HDL-C, IL-6 and CTRP-1 as covariates and with or without CHD as dependent variables. With forward stepwise (conditional) method: Wald, the binary logistic regression analysis, showed that increases in CTRP-1 and IL-6 were powerful predictive factors for the occurrence of CHD (Table 5).
DISCUSSION Inflammation is a primary factor promoting the initiation of coronary plaques, their unstable progression and their final disruption,14 which together underlie the pathogenesis of ACS. Immune cells dominate early atherosclerotic lesions. Their effector molecules accelerate the progression of the lesions, and the activation of inflammation can elicit ACS, which is an acute stage of CHD, AMI and UAP.1 Patients with a more obvious vascular inflammatory response have a poorer outcome. It is now well established that adipose tissue is not only involved in energy storage but also functions as an endocrine organ that secretes various bioactive substances. Factors that are secreted by adipose tissue are collectively referred to as adipokines15,16 and function in a pro- or anti-inflammation capacity in metabolic dysfunction. For instance, the TNF produced by monocytes and macrophages was identified as a proinflammatory product of adipose tissue17 that promotes the progression of diabetes, obesity and atherosclerosis. Recently, a new protein family of secreted adipokines, C1q/TNF-related protein (CTRP), was discovered. This family has been reported to provide a link between inflammation and metabolism.4 Each CTRP family member has its own unique tissue expression profile and function in regulating glucose and/or fat metabolism.18 As a family member of CTRP, fat tissue CTRP-1 expression was in vivo stimulated by rosiglitazone, a drug with anti-inflammatory properties and a high-affinity ligand for the adipogenic transcription factor peroxisome proliferator-activated receptor.5 In addition, IL-1b strongly upregulates CTRP-1 in the epididymal fat of SD rats, an effect that is, enhanced by TNF-a,7 both of which are induced by LPS. These data indicate that adipose tissue CTRP-1 expression is induced in inflammation. Whereas SVC is a major source of CTRP-1, and LPS-mediated upregulation in fat might also be related to enhanced synthesis in macrophages, whether higher CTRP-1 expression is associated with increased adipocyte and/or SVC synthesis or simply reflects altered SVC composition in obesity has not yet been determined. These findings suggest that CTRP-1
TABLE 3. Comparisons of plasma CTRP-1 and IL-6 in single, double and triple-vessel diseases n CTRP-1, ng/L 11.58 6 1.21a,b,c 12.93 6 1.62a 13.38 6 1.72a 9.90 6 0.57 ,0.01 F 5 40.60
IL-6, ng/L 119.02 159.99 209.94 46.92
(73.89–178.61)a,c,d (80.77–249.39)a,c (202.25–239.01)a (36.38–57.61) ,0.01 x2 5 72.50
Copyright Ó 2015 by the Southern Society for Clinical Investigation. Unauthorized reproduction of this article is prohibited.
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Singe-vessel lesion group, n (%) Double-vessel lesion group, n (%) Triple-vessel lesion group, n (%) Control group P
26 16 29 30
Data are expressed as mean 6 SD or median (interquartile range). a P , 0.01 in comparison with control group. b P , 0.01 in comparison with double-vessel group. c P , 0.01 in comparison with triple-vessel group. d P , 0.05 in comparison with double-vessel group. CTRP-1, C1q/TNF-related protein-1; IL-6, interleukin 6.
Tang et al
TABLE 4. Correlation analysis of circulating CTRP-1and IL-6 levels with cardiac risk factors CTRP1 IL-6
2
BMI, kg/m SBP, mm Hg DBP, mm Hg Heart rate/min TC, mmol/L TG, mmol/L LDL-C, mmol/L HDL-C, mmol/L hs-CRP, mg/dL CTRP-1 IL-6
r
P
r
P
20.043 20.169 0.122 0.207 0.018 0.148 0.105 20.432 0.520 — 0.667
0.703 0.131 0.276 0.063 0.873 0.188 0.353 ,0.01 ,0.01 — ,0.01
20.094 20.075 0.073 0.241 20.154 0.111 0.007 20.514 0.546 0.667 —
0.403 0.506 0.517 0.03 0.171 0.324 0.947 ,0.01 ,0.01 ,0.01 —
BMI, body mass index; CTRP-1, C1q/TNF-related protein-1; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.
is increased in obesity and that its upregulation might be related to the inflammatory aspect of this condition.3 In our study, CTRP-1 expression was much higher in the ACS group compared with the SAP or control groups, which suggests that CTRP-1 may function as an inflammatory agent in the acute phase of CHD and, as such, impact the stability of atheromatous plaque. Recently, Peterson et al19 demonstrated that CTRP-1 could act through the activation of AMP-activated protein kinase (AMPK) and inhibition of acetyl-CoA carboxylase in muscle to enhance fatty acid oxidation. Furthermore, AMPK has been noted to mediate preconditioning in isolated cardiomyocytes under hypoxic conditions.20 Increased AMPK activity likely compensates for the ischemic condition by upregulating GLUT4 translocation and enhancing fatty acid oxidation.21,22 Furthermore, Chalupova et al reported that circulating CTRP-1 levels were elevated in patients with metabolic syndrome compared with healthy people. In addition, CTRP-1 is correlated with glucose levels, HbA1c and BMI in metabolic syndrome patients,23 indicating that CTRP-1 is intimately linked to cardiovascular risk factors. In our study, we explored the correlations between the levels of CTRP-1 and cardiacrelated factors, and we found that CTRP-1 was negatively correlated with the cardiac protective factor HDL-C, which may provide additional evidence that CTRP-1 is closely related to cardiac risk factors. Simultaneously, the CTRP-1 concentrations were much higher in the groups with coronary vessel lesions (single, double or triple) compared with the healthy control group. And there was a significant difference between the single-vessel lesion group and double-vessel or triple-vessel groups, which may reflect a degree of coronary vessel stenosis.
Moreover, Lasser et al8 demonstrated that CTRP-1 could specifically bind to fibrillar collagen type I and block collageninduced platelet activation and aggregation. These observations indicate that CTRP-1 may play a role in the process of thrombosis, a key contributor to the development and aggravation of CHD. In this study, IL-6 cytokines and their signaling events were shown to contribute to both atherosclerotic plaque development and plaque destabilization through a variety of mechanisms.24 IL-6 is the eponym of an entire cytokine family, including molecules such as IL-11, IL-27, leukemia inhibitory factor, oncostatin M and several others.25 The expression of IL-6, a major determinant of the production of acute-phase proteins, is increased in a number of clinical conditions, such as tissue injury, infections, malignant neoplasms, ischemic diseases and trauma.26 One clinical trial, a case-control study of 294 patients with clinical ACS (group I) and clinically SAP (group II), showed the median IL-6 levels to be significantly higher in group I than in group II (P , 0.05).27 Experimentally, treatment with recombinant IL-6 was found to exacerbate the degree of atherosclerosis in atherosclerosis-prone ApoE-deficient mice and was accompanied by increased levels of other proinflammatory cytokines.28 Furthermore, it has been reported that trans-IL-6 receptor binding may alter intracellular signaling. Blocking of IL-6 receptor binding may be pathogenic in AMI, as IL-6 and the soluble IL-6 receptor were elevated, whereas IL-6R mRNA was decreased, in patients with AMI when compared with patients with stable CAD and controls.29 Using multivariable-adjusted Cox proportional hazards regression analyses in a prospective cohort study of 718 patients with CAD, the authors found that serum IL-6 was significantly associated with all-cause cardiovascular mortality.30 In this study, we demonstrated that IL-6 concentrations were significantly increased in patients with ACS or SAP compared with the healthy subjects. The IL-6 concentration in the triplevessel lesions was significantly higher compared with single- or double-vessel lesions, as well as a higher expression in the double-vessel lesion group compared with the single-vessel lesion group. This result indicated that IL-6 acted as an inflammatory factor and may be useful in evaluating the severity of CHD from the perspective of both the evolution of CHD and the degree of coronary artery stenosis. In addition, we conducted a correlation analysis on the levels of CTRP-1 and IL-6 with all cardiac-related factors that were evaluated in our study, including BMI, SBP, DBP, heart rate, TC, TG, LDL-C, HDL-C and hs-CRP. We found that the concentrations of both CTRP-1 and IL-6 were negatively correlated with HDL-C, whereas the levels of CTRP-1 and IL-6 were positively correlated with hs-CRP. We also detected a positive correlation between concentrations of CTRP-1 and IL-6 in patients with CHD. Although the detailed mechanism of how the cytokines interact is still unclear, there are 2 possible explanations for these correlations. First, atherosclerosis is a complex chronic inflammatory and metabolic disease that is, due to the interaction of several cellular components of the immune system involving a multitude of immune cell subsets
TABLE 5. Logistic regression analysis for the detection of independent relationship with the occurrence of CHD Variables b SE Wald x2 OR 95% CI Constant CTRP-1 IL-6
223.98 1.88 0.06
6.86 0.63 0.20
12.23 8.95 9.49
0.0 6.57 1.06
— 1.91–22.54 1.02–1.11
P ,0.01 0.003 0.002
CI, confidence interval; CHD, coronary heart disease; CTRP-1, C1q/TNF-related protein-1; IL-6, interleukin 6; OR, odds ratio; SE, standard error.
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Expressions of CTRP-1 and IL-6 in CHD
together with soluble molecules. This complex process leads to thickening of the arterial wall.31 CTRP-1 is secreted by stromal vascular SVC composed of adipose-tissue macrophages, and its expression is stimulated by TNF-a and IL-1b, which are both the inflammatory mediators that contribute to the process of CHD.7,32,33 In human coronary atherosclerotic plaques, IL-6 seems to stimulate macrophages and regulate the expression of adhesion molecules and other cytokines such as IL-1b and TNF-a, potentially enhancing the inflammatory reaction.10 Therefore, IL-6 may enhance the expression of CTRP-1, further adding to the inflammation associated with atherosclerotic plaques. Both CTRP-1 and IL-6 were positively correlated with the inflammatory factor hs-CRP, further supporting the role of CTRP-1 and IL-6 as inflammatory mediators. Second, CTRP1 is an adipocytokine associated with metabolic syndrome and obesity, which have similar risk factors as cardiovascular disease.23 IL-6 is also produced in the adipose tissue, linking obesity to a state of chronic low-level inflammation as a potential trigger for cardiovascular and metabolic diseases.24 In this study, both CTRP-1 and IL-6 were negatively correlated with the protective factor HDL-C, suggesting that CTRP-1 and IL-6 may both play an important role in cardiac and metabolic risk factors that influence the development of atherosclerosis. Finally, these 2 factors interact and thereby amplify the development of atherosclerosis, which might explain why both CTRP-1 and IL-6 are more highly expressed in the plasma of patients compared with the healthy group. The progression of CHD is the result of unstable coronary atherosclerotic plaques and the stenosis of coronary arterial atherosclerosis, both of which are closely linked with unbalanced inflammatory cytokines. We have described the plasma levels of CTRP-1 and IL-6 in CHD of different levels of severity. Dysregulated secretion of CTRP-1 and IL-6 is speculated to be involved in the pathogenesis of CHD, and the variation in CTRP-1 and IL-6 expression in CHD suggests that these proteins might be useful biomarkers of disease severity. In addition, in the logistic analysis for the detection of independent factors associated with the occurrence of CHD, we found that CTRP-1 and IL-6 may also play roles in predicting the development of CHD. Some limitations of the study must be considered. First, the study was conducted in a single center, and the number of subjects was small. Furthermore, plasma levels of CTRP-1 and IL-6 were not measured at different time points; thus, changes over time or potential fluctuations due to various medications were not examined. In addition, mechanisms through which CTRP-1 could influence the stability of coronary atherosclerotic plaques and the process of coronary arterial atherosclerosis as well as possible ischemia/reperfusion through the AMPK pathway are worthy of exploration. Hence, larger clinical trials, longer follow-up and research to uncover underlying mechanisms are needed to validate the findings of this study. In conclusion, patients with CHD (ACS or SAP) have significantly increased circulating concentrations of CTRP-1 and IL-6. This study suggests that the variation in plasma CTRP-1 and IL-6 concentrations may play an important role in reflecting the degree of inflammation in CHD and the severity of coronary arterial atherosclerosis. The combined determination of CTRP-1 and IL-6 may play a role in predicting the development of CHD. REFERENCES 1. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685–95.
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