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Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease
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JJCC-1387; No. of Pages 8 Journal of Cardiology xxx (2016) xxx–xxx
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Original article
Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease Kadri Murat Gurses (MD, PhD)a, Fusun Ozmen (MD, PhD)b, Duygu Kocyigit (MD)c, Nilgun Yersal (MSc)a, Elif Bilgic (MD)a, Erkan Kaya (MD)d, Cagla Zubeyde Kopru (PhD)e, Tolga Soyal (MD)f, Suat Doganci (MD)g, Lale Tokgozoglu (MD)c, Petek Korkusuz (MD, PhD)a,* a
Department of Histology and Embryology, Hacettepe University Faculty of Medicine, Ankara, Turkey Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey c Department of Cardiology, Hacettepe University Faculty of Medicine, Ankara, Turkey d Department of Cardiovascular Surgery, Gaziantep University Sahinbey Research and Application Hospital, Gaziantep, Turkey e Department of Nanotechnology and Nanomedicine, Hacettepe University Graduate School of Science and Engineering, Ankara, Turkey f Department of Cardiovascular Surgery, Medicana International Ankara, Turkey g Department of Cardiovascular Surgery, Gulhane Military Medical Academy, Ankara, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2016 Received in revised form 25 August 2016 Accepted 29 August 2016 Available online xxx
Background: Inflammatory activity originating from the epicardial adipose tissue (EAT) may have a role in coronary artery disease (CAD) pathogenesis. The relationship between macrophage infiltration, polarization in the EAT, and netrin-1 gene expression was investigated. Methods: Macrophage infiltration and polarization were examined by immunohistochemical methods and expression levels of netrin-1, Unc5b, and cytokines related with M1-macrophage subtype (IL-12 and IL-18) were determined by quantitative polymerase chain reaction in subcutaneous and epicardial adipose tissue obtained from patients undergoing coronary artery bypass grafting and non-coronary cardiac surgery. Results: CAD patients had higher CD68+ (p = 0.005) and CD11c+ (p < 0.001) macrophage count in EAT when compared to the controls. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the controls (p = 0.008). CAD patients had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p < 0.001, p < 0.001, and p = 0.006 respectively). Increased macrophage infiltration and polarization were associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (p < 0.05). Conclusions: Findings suggest a link between enhanced netrin-1 expression in EAT and macrophage infiltration and polarization in patients with CAD. ß 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Keywords: Netrin-1 Epicardial adipose tissue Macrophage polarization Coronary artery disease
Introduction Epicardial adipose tissue (EAT) is located between the myocardium and the serous layer of the pericardium. It is in close proximity with the coronary arteries [1]. It shares the same origin with the omental and mesenteric adipose tissue, and therefore is able to secrete cytokines and adipokines similar to the
* Corresponding author at: Department of Histology and Embryology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey. Fax: +90 312 310 05 80. E-mail address: [email protected] (P. Korkusuz).
other visceral adipose tissues [2]. Although it makes up a small part of the total fat in the whole body (approximately 0–3%), EAT has been shown to have a substantial impact on coronary arteries and myocardium. Due to the lack of fascia between coronary arteries and EAT, adipokines and cytokines secreted from EAT have paracrine effects [3–5]. A shift in macrophage phenotype to M1 macrophages was reported in EAT samples obtained from obese patients with coronary artery disease (CAD) [6,7]. Netrin-1 is a laminin-like protein that functions as a guidance protein in the course of neuronal migration during embryonic development [8]. The role of netrin-1 expression in macrophage infiltration and polarization in various tissues other than EAT have
http://dx.doi.org/10.1016/j.jjcc.2016.08.016 0914-5087/ß 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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been evaluated in previous studies [9,10]. Its receptor Unc5b, which is known to have anti-chemotactic functions, was reported to be expressed in the heart, the kidneys, and the lungs [10]. It is also highly expressed in human granulocytes, monocytes, and lymphocytes [10]. In a recent study, increased netrin-1 expression in omental adipose tissue samples obtained from subjects with obesity has been suggested to have a role in chronic inflammation and insulin resistance, acting via Unc5b receptors [6]. Whether netrin-1 is expressed in EAT of patients with CAD and has a role in enhanced macrophage infiltration and polarization has not been evaluated to date. Research questions of this study were whether (1) macrophage infiltration and polarization in EAT would differ between CAD patients and controls and (2) netrin-1 and Unc5b expressions in EAT were related to macrophage infiltration and polarization. Assessment of netrin-1, Unc5B, and cytokines related to M1macrophage subtype (IL-12, IL-18) expression in epicardial and subcutaneous adipose tissue (SAT) samples belonging to patients with CAD undergoing coronary artery bypass grafting (CABG) was aimed. We furthermore aimed to determine the relationship between netrin-1 expression and macrophage infiltration and polarization in the same cohort using immunohistochemistry and polymerase chain reaction (PCR) techniques. Materials and methods Design A cross-sectional clinical study was designed. Dr Abdurrahman Yurtaslan Ankara Training and Research Hospital Clinical Research Ethics Committee approved the study (Project number: 2014-7/ 104). Informed consent was obtained for the use of the human tissues. The study conformed to the ethical principles contained in the Declaration of Helsinki.
steroid or immunomodulatory drug use, history of chemotherapy or radiotherapy were excluded. A total of 32 patients [71.88% male, 57.00 (53.00–66.75) years] who underwent cardiac surgery were included in the study. Baseline clinical and laboratory parameters, including age and gender, did not differ between groups (Table 1). Immunohistochemical staining Adipose tissue samples were fixed in formalin and paraffinembedded tissue blocks were prepared. Consecutive 3–5 mm-thick sections were cut from each paraffin sample. A microwave oven was used for antigen retrieval and the procedure was performed in trisodium citrate buffer (C-8532, Sigma, Munich, Germany) (0.1 M, pH = 6) in 10 min. Primary antibodies against anti-human CD68 (1:500, HPA048982-Atlas Antibodies, Stockholm, Sweden), CD11c (1:100, HPA004723-Atlas Antibodies), CD206 (1:100, HPA004114Atlas Antibodies), netrin-1 (1:100, HPA056419-Atlas Antibodies) and Unc5b (1:100, HPA011141-Atlas Antibodies) were utilized for immunohistochemical staining of 5 mm-thick consecutive sections. Following collection in 0.01 M phosphate buffer solution (PBS), sections were incubated in peroxidase block (EXTRA3,1KT, Sigma) for 30 min at room temperature. Sections were then placed in chromogen 3,30 -diaminobenzidine (DAB) (D3939-1SET-Sigma) solution for 5 min and washed with PBS. Nuclear background staining was done with hematoxylin. Human tonsil and large intestine were used as positive control tissue. Primary antibody step was omitted and non-specific immunoglobulin was applied as negative control. Immune-labeled cells were counted on at least 5 sites per section at 400 magnification and the average was recorded by two blinded investigators according to the literature [12]. Real-time PCR
Study population Eighteen patients with CAD who underwent elective CABG in the Department of Cardiovascular Surgery were enrolled in this study. Fourteen patients who underwent heart valve surgery were included as the control group. All patients had significant proximal left anterior descending (LAD) artery stenosis. EAT samples were obtained from the anterior wall of the left ventricle, adjacent to the diseased segment of LAD artery. SAT samples were obtained from the sternal subcutaneous fat. Gensini score was calculated in the patient group to determine the severity of CAD [11]. Patients with atrial arrhythmias, prior and/or current cardiac inflammatory diseases (i.e. myocarditis, pericarditis, and pericardial effusion), hematologic diseases, rheumatologic diseases,
Total RNA was extracted from EAT and SAT specimens using an RNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the instructions. Tissue lysis was performed with Mini Bead Beater-8 (Biospec, Bartlesville, OK, USA). Purity and concentration of RNAs were measured with the NanoDrop 1000 spectrophotometer (Thermo, Waltham, MA, USA). cDNA was subsequently synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo) with 260 ng of total RNA isolated for each sample. The quantitative real-time (RT)-PCR (qRT-PCR) assays were performed with the Real-Time Ready Single Assay (Roche Applied Science, Pleasanton, CA, USA) kit using LightCycler1 480 probes Master (Roche Applied Science), and the gene expression levels of beta-actin, netrin-1, Unc5b, IL-12, and IL-18 were quantified. The
Table 1 Baseline clinical and laboratory parameters of the study population (n = 32).
Age (years) Gender: male, n (%) BMI (kg/m2) Hypertension, n (%) Diabetes mellitus, n (%) Smoking, n (%) White blood cell count (103/mL) LDL cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglyceride (mg/dL)
Study population (n = 32)
CAD (n = 14)
CAD+ (n = 18)
57.00 (53.00–66.75) 23 (71.88) 22.55 (20.81–26.24) 15 (46.88) 5 (15.63) 11 (34.38) 6.90 (5.83–9.13) 138.00 (106.25–164.00) 45.00 (40.00–54.53) 114.00 (91.50–183.25)
57.00 (53.00–68.50) 9 (64.29) 21.36 (20.80–26.07) 6 (42.86) 2 (14.29) 4 (28.57) 7.10 (5.85–8.83) 112.00 (93.75–151.50) 48.50 (42.63–55.28) 111.00 (84.75–133.75)
58.00 (51.00–67.00) 14 (77.78) 23.26 (21.20–26.72) 9 (50.00) 3 (16.67) 7 (38.89) 6.80 (5.70–9.35) 148.00 (117.50–166.75) 44.00 (40.00–53.50) 138.50 (92.50–231.00)
p-Value 0.639 0.423 0.319 0.735 1.000 0.712 1.000 0.107 0.338 0.180
BMI, body mass index; CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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qPCR reactions were performed in a volume of 20 mL. The cycling conditions were as follows: 95 8C for 10 s, 60 8C for 30 s, and 72 8C for 1 s for 45 cycles, with initial melting at 95 8C for 10 min. PCR threshold cycle number (CT) was used to calculate relative expression levels for each tissue and control sample using the DD D D following formulas: 2 Ct = 2( Ct sample Ct control) and DDCt [(Ct sampleCt reference)(Ct controlCt reference)] 2 =2 [13,14]. DCT represents the difference in CT values between the target and reference gene beta-actin. RT-PCR was performed twice and average CT values were obtained. Statistical analysis Homogeneity of variances and normal distribution were tested with Shapiro–Wilk test. Mean standard deviation and median (interquartile range defined as 25th–75th percentiles) were used to express normally and skewed continuous parameters, respectively. Frequencies and percentages were used to describe categorical parameters and were compared with chi-square test. Non-normally distributed parameters were compared with Bonferroni corrected Mann–Whitney U test and Wilcoxon test for paired samples. Spearman correlation analysis was performed to test the relation between non-normally distributed variables. Independent predictors for CAD were determined with binomial regression analysis. Analyses were performed using SPSS statistical software (version 21.0; SPSS Inc., Chicago, IL, USA). A two-tailed p < 0.05 was considered statistically significant.
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Results Macrophage infiltration and polarization in EAT Macrophage infiltration was increased in EAT of CAD patients. CAD patients had significantly higher CD68+ macrophage count in EAT when compared to the control group (p = 0.005). CD11c+ macrophage count was also significantly higher in patients with CAD when compared to that of the control group (p < 0.001). CD206+ macrophage count did not differ between groups. Although CD68+/total nuclei count ratio, which can be considered as a more objective parameter of macrophage infiltration, tended to be higher in EAT of the patient group, it did not reach statistical significance (p = 0.052). In the patient group, CD68+ macrophage infiltration was higher in the EAT to that of SAT (p = 0.001). CD68+/ total nuclei count ratio, CD11c+ and CD206+ macrophage counts were also significantly higher in the EAT of the patient group when compared to SAT of the patient group (p = 0.002, p = 0.003 and p = 0.002, respectively). None of the parameters related to the macrophage infiltration differed between EAT and SAT in the control group (Fig. 1). Macrophage polarization shifted into a pro-inflammatory profile in EAT of CAD patients. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1-macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the control group (p = 0.008). CD11c+/CD206+ macrophage ratio was also significantly higher in the EAT when compared to the
Fig. 1. Representative micrographs showing macrophages exhibiting CD68 immunoreactivity on (a) epicardial adipose tissue (EAT) of coronary artery disease (CAD) patients (n = 18), (b) subcutaneous adipose tissue (SAT) of CAD patients (n = 18), and (c) EAT of control group (n = 14). CD68-positive macrophages (asterisk) are mostly located at the perivascular region. Scale bar = 50 mm. CD68-positive cell count (d) and CD68/total nuclei count ratio (e) in EAT and SAT of patient and control groups are shown in the lower panel. # sign denotes statistical significance (p < 0.05).
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 2. (a) Representative micrographs showing CD11c (upper panel) and CD206 immune-positive cells (lower panel) in epicardial adipose tissue (EAT) of coronary artery disease (CAD) patients (n = 18), subcutaneous adipose tissue (SAT) of CAD patients (n = 18), and EAT of control group (n = 14). Scale bar = 50 mm. (b) CD11c+/CD206+ macrophage ratio in EAT and SAT of patient and control groups is shown. # sign denotes statistical significance (p < 0.05).
SAT of the patient group (p = 0.023). None of the macrophage polarization parameters differed between EAT and SAT in the control group (Fig. 2). Netrin-1, Unc5b, IL-12, and IL-18 expression in EAT and SAT Expression of netrin-1, Unc5b, and cytokines related to M1macrophage subtype (IL-12, IL-18) in EAT were increased in CAD patients. Patients with CAD had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p < 0.001, p < 0.001, and p = 0.006 respectively) (Fig. 3). None of the other parameters, including diabetes mellitus, hypertension, smoking, obesity [body mass index (BMI) 25 kg/ m2], or hyperlipidemia [low-density lipoprotein (LDL) 130 mg/ dL] were associated with the gene expression of netrin-1, Unc5b, IL12, and IL-18 in EAT (Table 2). Furthermore, no correlation was detected between netrin-1 gene expression and age, white blood cell count, and levels of LDL-cholesterol, high-density lipoprotein cholesterol or triglyceride. Netrin-1 expression was positively correlated with BMI (r = 0.287, p = 0.111) and Gensini score
(r = 0.387, p = 0.113); however, these did not reach statistical significance. Netrin-1 gene expression in EAT was positively correlated with gene expression of its receptor Unc5b in a statistically significant way (r = 0.590, p < 0.001). Netrin-1 gene expression was also positively and significantly correlated with IL-12 (r = 0.365, p = 0.040) and IL-18 (r = 0.386, p = 0.029) gene expression. Immunohistochemical analysis demonstrated netrin-1 and Unc5b staining on perivascular macrophages and endothelium in the adipose tissue. Both macrophages and endothelial cells exhibited moderate-to-high cytoplasmic immune reaction with netrin-1 and mild-to-moderate cytoplasmic immune reaction with Unc5b. Number of macrophages that were labeled with netrin-1 [15.00 (11.00–17.00) vs. 12.00 (10.75–13.00), p = 0.004] and Unc5b [9.00 (6.50–11.50) vs. 5.50 (5.00–6.50), p = 0.027] were significantly higher in EAT of the patient group when compared to that of the control group. Number of macrophages exhibiting netrin-1 (r = 0.609, p = 0.001) and Unc5b (r = 0.488, p = 0.010) immunoreactivity was correlated with their gene expression profile on qPCR in EAT (Fig. 4). Relationship between gene expression of netrin-1, Unc5b, IL-12, IL-18, and macrophage infiltration and polarization in EAT Netrin-1 and Unc5b expressions were found to be related with macrophage infiltration and polarization in EAT. Increased CD68+/ total nuclei count ratio (CD68+/total nuclei count ratio 0.11) was associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (Fig. 5a). Increased CD11c+/CD206+ macrophage ratio (CD11c+/CD206+ macrophage ratio 0.55) was found to be associated with higher gene expression of netrin-1, Unc5b, and IL-18 (Fig. 5b). Netrin-1 gene expression in EAT was also found to be positively correlated with CD68+/total nuclei count ratio in a statistically significant way (r = 0.649, p < 0.001). CD11c+/CD206+ macrophage count ratio was positively correlated (r = 0.369, p = 0.058) with netrin-1 gene expression in EAT; however, it did not reach statistical significance. Determination of predictors of CAD
Fig. 3. Netrin-1, Unc5b, IL-12, and IL-18 gene expression in epicardial adipose tissue of the patient (n = 18) and control (n = 14) groups. # sign denotes statistical significance (p < 0.05).
In the multivariate regression analysis model including LDLcholesterol and triglyceride levels, netrin-1 gene expression was found to be independently associated with presence of CAD (OR: 1.520, 95% CI: 1.123–2.058, p = 0.007).
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Table 2 Relationship between gene expression of netrin-1, Unc5b, IL-12, and IL-18, and baseline clinical and laboratory parameters. 2
DDCT
netrin-1
p-Value
DDCT
2
Unc5b
p-Value
2
DDCT
IL-12
p-Value
2
DDCT
IL-18
0.37 (0.13–1.20) <0.001* 0.69 (0.26–1.28) <0.001* 1.56 (0.65–2.14) 0.587 1.21 (0.43–1.85) CAD (n = 14) 2.98 (1.49–4.02) 1.48 (0.77–2.84) 2.77 (1.07–5.79) CAD+ 4.77 (2.13–16.01) (n = 18) 0.285 1.64 (0.82–3.15) 0.614 1.18 (0.66–2.08) 0.136 1.80 (0.90–4.04) DM 1.78 (0.28–6.21) (n = 27) 1.50 (0.25–6.89) 2.05 (1.62–2.87) 2.78 (0.44–5.12) 3.40 (1.80–12.99) DM+ (n = 5) 0.331 1.33 (0.52–3.31) 0.502 1.69 (0.72–2.59) 0.941 1.35 (0.77–2.78) 1.78 (0.37–4.77) HT (n = 17) 1.89 (0.99–3.05) 1.42 (0.75–2.06) 2.76 (1.02–4.94) 3.01 (0.88–14.27) HT+ (n = 15) 0.389 1.50 (0.91–2.79) 0.785 1.55 (0.86–2.82) 0.194 1.82 (0.84–4.76) Smoking 2.51 (0.67–10.29) (n = 21) 2.53 (0.36–3.61) 0.99 (0.54–1.95) 1.13 (0.75–2.82) Smoking+ 1.48 (0.28–3.32) (n = 11) 0.710 1.89 (0.77–3.47) 0.655 1.69 (0.66–2.08) 0.710 1.80 (1.06–4.94) 1.08 (0.24–14.27) LDL <130 mg/dL (n = 15) 1.45 (0.62–3.10) 1.18 (0.77–2.59) 1.82 (0.74–3.70) LDL 130 mg/dL 2.22 (1.48–3.91) (n = 17) 0.254 1.33 (0.52–2.72) 0.104 1.69 (0.86–2.22) 0.785 1.82 (0.96–3.70) BMI <25 kg/m2 1.87 (0.37–3.91) (n = 21) 2 2.67 (1.32–5.24) 1.41 (0.66–2.88) 1.63 (0.72–4.94) 3.32 (0.88–33.36) BMI 25 kg/m (n = 11) BMI, body mass index; CAD, coronary artery disease; DM, diabetes mellitus; HT, hypertension; IL, interleukin; LDL, low-density lipoprotein. * sign denotes statistical significance (p < 0.05).
Discussion This is the first study in the literature demonstrating enhanced netrin-1 and Unc5b expression in the EAT of patients with CAD, and the link between their expression and macrophage infiltration and polarization in EAT. Our study has shown that CD68+ macrophage infiltration was increased in EAT of patients with CAD when compared to the control group. This finding is compatible with the results of prior studies reporting inflammation in the EAT in patients with CAD, reflected by increased CD68+ macrophage infiltration [7,15]. On the contrary, Kitagawa et al. [16] have shown that, although more prominent in patients with CAD, CD68+ macrophage infiltration in EAT did not differ in a statistically significant way among patients who underwent coronary and non-coronary cardiac surgery. When macrophage subtypes are taken into consideration, our study has revealed that only CD11c+, but not CD206+, macrophage infiltration was significantly higher in EAT of the patient group when compared to the controls. On the other hand, the single previous study that evaluated macrophage subtypes in EAT in CAD, Hirata et al. [7] reported statistically significantly increased levels of both CD11c+ and CD206+ macrophages when compared to the control group. Increase in macrophage infiltration in the EAT of CAD patients was also confirmed in our study by comparing CD68+ macrophage infiltration in EAT and SAT of the patient group. Subjects with CAD had significantly higher CD68+ macrophage count and CD68+ macrophage/total nuclei count ratio in the EAT when compared to SAT. Similar findings were obtained in prior studies [7,16]. Our study also revealed that CD11c+ and CD206+ macrophage counts were also significantly higher in EAT when compared to SAT in subjects with CAD, in concordance with findings reported by Hirata et al. [7]. Our study demonstrated a shift to pro-inflammatory M1macrophage phenotype in EAT of the patient group when compared to the control group, reflected by increased CD11c+/ CD68+ and CD11c+/CD206+, and decreased CD206+/CD68+ macrophage ratios. M1/M2-macrophage ratio was also found to be higher in the EAT when compared to SAT in the patient group. Hirata et al. [7] have reported similar findings, suggesting that
p-Value 0.006*
1.000
0.370
0.785
0.502
0.611
pro-inflammatory macrophages are more dominant in EAT when both compared to the control group’s and to the patients’ SAT. Findings of our study are unique to demonstrate higher netrin-1 and Unc5b gene expression levels in EAT of subjects with CAD evaluated for the first time in the literature. Gene expression of Unc5b in EAT, which is considered to be the receptor involved in netrin-1’s effects on cardiovascular system, was found to be correlated with netrin-1 gene expression. Furthermore, netrin-1 expression was found to be associated with CAD presence independent of other cardiovascular risk factors. Although positively correlated with Gensini score, which is used to determine the severity of CAD, the correlation between Gensini score and netrin-1 expression failed to reach statistical significance possibly due to the relatively small study population. Studies have shown a wide spectrum of effects of netrin-1 on various tissues, including lungs, kidneys, and endothelial cells [10,17–21]. A single study that evaluated netrin-1 expression in the adipose tissue focused on netrin-1 expression in the omental adipose tissue and reported that it was higher in obese mice and humans, but not in non-obese, and was associated with macrophage retention [22]. In that study, upregulation of netrin-1 synthesis in macrophages with palmitate or exposing adipose tissue-located macrophages of non-obese mice to netrin-1 were shown to inhibit both migration response to chemokine (C–C motif) ligand 19 (CCL19), and dendritic cell and macrophage migration to the peripheral lymph nodes [22]. Furthermore, the aforementioned impaired migration response to CCL19 has been shown to be reversed with Unc5b blockage [22]. These findings have suggested that synthesis of netrin-1 by adipose tissue-located macrophages may lead to accumulation of macrophages and eventually lead to chronic, metabolic dysfunction originating from the adipose tissue. In our study, we demonstrated a positive correlation between netrin-1 expression and BMI, but it failed to reach statistical significance. Further studies are required to elucidate whether netrin-1 expression in EAT of subjects with CAD is independent of peripheral obesity. The role of netrin-1 in atherosclerosis has been investigated in a few studies [23]. Although netrin-1 has been shown to function as a
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 4. (a) Representative micrographs showing netrin-1 and Unc5b-positive cells (denoted by asterisk). Scale bar = 50 mm. (b) Correlation graphs for netrin-1 and Unc5b gene expression and immunohistochemical labeling are shown. EAT, epicardial adipose tissue; SAT, subcutaneous adipose tissue.
‘‘barrier’’ in other tissues that prevent infiltration of leukocytes, studies in atherosclerotic plaque have demonstrated pro-atherosclerotic properties related to netrin-1 expression by the foam cells. Recombinant netrin-1 secreted from in vitro foam cells has been reported to inhibit the migration response of macrophages to CCL19 [24,25]. van Gils et al. [26] have shown that human and mice atherosclerotic plaque foam cells expressed netrin-1 and Unc5b and selective deletion of netrin-1 in bone marrow of Lldr knockout mice resulted in smaller atherosclerotic lesion and decreased inflammatory activity within the plaque [26]. These findings have promoted the hypothesis that netrin-1 may act as a player in progression of atherosclerotic plaque via inhibition of cell regression and promotion of smooth muscle cell accumulation. Recently, Ramkhelawon et al. [27] have demonstrated that netrin-1 and Unc5b were expressed in the hypoxic regions of the mouse and human atherosclerotic plaques. Authors have demonstrated that netrin-1 and Unc5b expressions were induced with
hypoxia-inducible factor 1-alpha (HIF-1a), as well as oxidized LDL or oxidative stress [27]. This expression pattern has been attributed to hypoxia leading to impaired macrophage regression and enhanced macrophage survival [27]. Nevertheless, apart from the effects of netrin-1 on macrophage retention and survival which may be involved in enhanced inflammation in the EAT that aggravates coronary atherosclerosis and cardiac ischemia, it is also likely that inflammatory pathways that are associated with cardiac ischemia may lead to inflammation of EAT via paracrine actions, per se. These inflammatory pathways may be linked with various endogenous ligands that are called ‘‘danger-associated molecular patterns’’, and are known to be released upon injury and modulate inflammation in cardiac ischemia [28]. Our study has shown for the first time that increased macrophage infiltration was associated and positively correlated with netrin-1 expression in EAT. Netrin-1 expression in EAT was also greater in patients with predominantly M1-macrophage
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 5. Association of netrin-1, Unc5b, and M1-macrophage-related cytokine expression with (a) CD68/total nuclei count and (b) CD11c+/CD206+ macrophage ratio. # sign denotes statistical significance (p < 0.05).
phenotype. M1-macrophage-related cytokine IL-18 gene expression was found to be significantly higher in the EAT of the patient group when compared to the control group, which was associated with the predominancy of M1-macrophages. Although a similar association was not observed with IL-12 gene expression, netrin-1 expression was found to be correlated with both IL-12 and IL-18, suggesting another pro-inflammatory effect of netrin-1 in the EAT. To date, investigations to intervene the inflammation in the visceral adipose tissue have primarily focused on insulin sensitization. Nevertheless, these approaches fail since they do not target the initiating factors. We have demonstrated the involvement of netrin-1-Unc5b signaling pathway in EAT-originating or EATaccompanying chronic inflammation in coronary atherosclerosis for the first time. Inhibition of the netrin-1-Unc5b pathway may attenuate the inflammation in the EAT and the coronary arteries. Thus, molecular therapy targeting this pathway in EAT may be beneficial in preventing coronary atherosclerosis or its progression. Study limitations Our study has some limitations. First, the sample size is relatively small. However, this may not be considered as a major limitation, since previous studies with homologous study design also had similar size of study population and our findings reached statistical significance. Second, our study reveals an association, but not a clear causal relationship. Knockout animal model and in vitro cell culture studies may be implemented to explain the causality. Conclusions In conclusion, our study has shown an increase in netrin-1 expression that could be linked with macrophage infiltration and polarization in the EAT of patients with CAD. Demonstration of the involvement of this signaling pathway in EAT-originating chronic inflammation leading to atherosclerosis may have therapeutic implications in the future. Funding source This work was funded by Hacettepe University Scientific Research Projects Coordination Unit (Project number: 014 D11 101 003-705).
Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment Information reported in the article was presented at EAS Congress 2016.
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Contents lists available at ScienceDirect
Journal of Cardiology journal homepage: www.elsevier.com/locate/jjcc
Original article
Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease Kadri Murat Gurses (MD, PhD)a, Fusun Ozmen (MD, PhD)b, Duygu Kocyigit (MD)c, Nilgun Yersal (MSc)a, Elif Bilgic (MD)a, Erkan Kaya (MD)d, Cagla Zubeyde Kopru (PhD)e, Tolga Soyal (MD)f, Suat Doganci (MD)g, Lale Tokgozoglu (MD)c, Petek Korkusuz (MD, PhD)a,* a
Department of Histology and Embryology, Hacettepe University Faculty of Medicine, Ankara, Turkey Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey c Department of Cardiology, Hacettepe University Faculty of Medicine, Ankara, Turkey d Department of Cardiovascular Surgery, Gaziantep University Sahinbey Research and Application Hospital, Gaziantep, Turkey e Department of Nanotechnology and Nanomedicine, Hacettepe University Graduate School of Science and Engineering, Ankara, Turkey f Department of Cardiovascular Surgery, Medicana International Ankara, Turkey g Department of Cardiovascular Surgery, Gulhane Military Medical Academy, Ankara, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2016 Received in revised form 25 August 2016 Accepted 29 August 2016 Available online xxx
Background: Inflammatory activity originating from the epicardial adipose tissue (EAT) may have a role in coronary artery disease (CAD) pathogenesis. The relationship between macrophage infiltration, polarization in the EAT, and netrin-1 gene expression was investigated. Methods: Macrophage infiltration and polarization were examined by immunohistochemical methods and expression levels of netrin-1, Unc5b, and cytokines related with M1-macrophage subtype (IL-12 and IL-18) were determined by quantitative polymerase chain reaction in subcutaneous and epicardial adipose tissue obtained from patients undergoing coronary artery bypass grafting and non-coronary cardiac surgery. Results: CAD patients had higher CD68+ (p = 0.005) and CD11c+ (p < 0.001) macrophage count in EAT when compared to the controls. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the controls (p = 0.008). CAD patients had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p < 0.001, p < 0.001, and p = 0.006 respectively). Increased macrophage infiltration and polarization were associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (p < 0.05). Conclusions: Findings suggest a link between enhanced netrin-1 expression in EAT and macrophage infiltration and polarization in patients with CAD. ß 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Keywords: Netrin-1 Epicardial adipose tissue Macrophage polarization Coronary artery disease
Introduction Epicardial adipose tissue (EAT) is located between the myocardium and the serous layer of the pericardium. It is in close proximity with the coronary arteries [1]. It shares the same origin with the omental and mesenteric adipose tissue, and therefore is able to secrete cytokines and adipokines similar to the
* Corresponding author at: Department of Histology and Embryology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey. Fax: +90 312 310 05 80. E-mail address: [email protected] (P. Korkusuz).
other visceral adipose tissues [2]. Although it makes up a small part of the total fat in the whole body (approximately 0–3%), EAT has been shown to have a substantial impact on coronary arteries and myocardium. Due to the lack of fascia between coronary arteries and EAT, adipokines and cytokines secreted from EAT have paracrine effects [3–5]. A shift in macrophage phenotype to M1 macrophages was reported in EAT samples obtained from obese patients with coronary artery disease (CAD) [6,7]. Netrin-1 is a laminin-like protein that functions as a guidance protein in the course of neuronal migration during embryonic development [8]. The role of netrin-1 expression in macrophage infiltration and polarization in various tissues other than EAT have
http://dx.doi.org/10.1016/j.jjcc.2016.08.016 0914-5087/ß 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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been evaluated in previous studies [9,10]. Its receptor Unc5b, which is known to have anti-chemotactic functions, was reported to be expressed in the heart, the kidneys, and the lungs [10]. It is also highly expressed in human granulocytes, monocytes, and lymphocytes [10]. In a recent study, increased netrin-1 expression in omental adipose tissue samples obtained from subjects with obesity has been suggested to have a role in chronic inflammation and insulin resistance, acting via Unc5b receptors [6]. Whether netrin-1 is expressed in EAT of patients with CAD and has a role in enhanced macrophage infiltration and polarization has not been evaluated to date. Research questions of this study were whether (1) macrophage infiltration and polarization in EAT would differ between CAD patients and controls and (2) netrin-1 and Unc5b expressions in EAT were related to macrophage infiltration and polarization. Assessment of netrin-1, Unc5B, and cytokines related to M1macrophage subtype (IL-12, IL-18) expression in epicardial and subcutaneous adipose tissue (SAT) samples belonging to patients with CAD undergoing coronary artery bypass grafting (CABG) was aimed. We furthermore aimed to determine the relationship between netrin-1 expression and macrophage infiltration and polarization in the same cohort using immunohistochemistry and polymerase chain reaction (PCR) techniques. Materials and methods Design A cross-sectional clinical study was designed. Dr Abdurrahman Yurtaslan Ankara Training and Research Hospital Clinical Research Ethics Committee approved the study (Project number: 2014-7/ 104). Informed consent was obtained for the use of the human tissues. The study conformed to the ethical principles contained in the Declaration of Helsinki.
steroid or immunomodulatory drug use, history of chemotherapy or radiotherapy were excluded. A total of 32 patients [71.88% male, 57.00 (53.00–66.75) years] who underwent cardiac surgery were included in the study. Baseline clinical and laboratory parameters, including age and gender, did not differ between groups (Table 1). Immunohistochemical staining Adipose tissue samples were fixed in formalin and paraffinembedded tissue blocks were prepared. Consecutive 3–5 mm-thick sections were cut from each paraffin sample. A microwave oven was used for antigen retrieval and the procedure was performed in trisodium citrate buffer (C-8532, Sigma, Munich, Germany) (0.1 M, pH = 6) in 10 min. Primary antibodies against anti-human CD68 (1:500, HPA048982-Atlas Antibodies, Stockholm, Sweden), CD11c (1:100, HPA004723-Atlas Antibodies), CD206 (1:100, HPA004114Atlas Antibodies), netrin-1 (1:100, HPA056419-Atlas Antibodies) and Unc5b (1:100, HPA011141-Atlas Antibodies) were utilized for immunohistochemical staining of 5 mm-thick consecutive sections. Following collection in 0.01 M phosphate buffer solution (PBS), sections were incubated in peroxidase block (EXTRA3,1KT, Sigma) for 30 min at room temperature. Sections were then placed in chromogen 3,30 -diaminobenzidine (DAB) (D3939-1SET-Sigma) solution for 5 min and washed with PBS. Nuclear background staining was done with hematoxylin. Human tonsil and large intestine were used as positive control tissue. Primary antibody step was omitted and non-specific immunoglobulin was applied as negative control. Immune-labeled cells were counted on at least 5 sites per section at 400 magnification and the average was recorded by two blinded investigators according to the literature [12]. Real-time PCR
Study population Eighteen patients with CAD who underwent elective CABG in the Department of Cardiovascular Surgery were enrolled in this study. Fourteen patients who underwent heart valve surgery were included as the control group. All patients had significant proximal left anterior descending (LAD) artery stenosis. EAT samples were obtained from the anterior wall of the left ventricle, adjacent to the diseased segment of LAD artery. SAT samples were obtained from the sternal subcutaneous fat. Gensini score was calculated in the patient group to determine the severity of CAD [11]. Patients with atrial arrhythmias, prior and/or current cardiac inflammatory diseases (i.e. myocarditis, pericarditis, and pericardial effusion), hematologic diseases, rheumatologic diseases,
Total RNA was extracted from EAT and SAT specimens using an RNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the instructions. Tissue lysis was performed with Mini Bead Beater-8 (Biospec, Bartlesville, OK, USA). Purity and concentration of RNAs were measured with the NanoDrop 1000 spectrophotometer (Thermo, Waltham, MA, USA). cDNA was subsequently synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo) with 260 ng of total RNA isolated for each sample. The quantitative real-time (RT)-PCR (qRT-PCR) assays were performed with the Real-Time Ready Single Assay (Roche Applied Science, Pleasanton, CA, USA) kit using LightCycler1 480 probes Master (Roche Applied Science), and the gene expression levels of beta-actin, netrin-1, Unc5b, IL-12, and IL-18 were quantified. The
Table 1 Baseline clinical and laboratory parameters of the study population (n = 32).
Age (years) Gender: male, n (%) BMI (kg/m2) Hypertension, n (%) Diabetes mellitus, n (%) Smoking, n (%) White blood cell count (103/mL) LDL cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglyceride (mg/dL)
Study population (n = 32)
CAD (n = 14)
CAD+ (n = 18)
57.00 (53.00–66.75) 23 (71.88) 22.55 (20.81–26.24) 15 (46.88) 5 (15.63) 11 (34.38) 6.90 (5.83–9.13) 138.00 (106.25–164.00) 45.00 (40.00–54.53) 114.00 (91.50–183.25)
57.00 (53.00–68.50) 9 (64.29) 21.36 (20.80–26.07) 6 (42.86) 2 (14.29) 4 (28.57) 7.10 (5.85–8.83) 112.00 (93.75–151.50) 48.50 (42.63–55.28) 111.00 (84.75–133.75)
58.00 (51.00–67.00) 14 (77.78) 23.26 (21.20–26.72) 9 (50.00) 3 (16.67) 7 (38.89) 6.80 (5.70–9.35) 148.00 (117.50–166.75) 44.00 (40.00–53.50) 138.50 (92.50–231.00)
p-Value 0.639 0.423 0.319 0.735 1.000 0.712 1.000 0.107 0.338 0.180
BMI, body mass index; CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
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qPCR reactions were performed in a volume of 20 mL. The cycling conditions were as follows: 95 8C for 10 s, 60 8C for 30 s, and 72 8C for 1 s for 45 cycles, with initial melting at 95 8C for 10 min. PCR threshold cycle number (CT) was used to calculate relative expression levels for each tissue and control sample using the DD D D following formulas: 2 Ct = 2( Ct sample Ct control) and DDCt [(Ct sampleCt reference)(Ct controlCt reference)] 2 =2 [13,14]. DCT represents the difference in CT values between the target and reference gene beta-actin. RT-PCR was performed twice and average CT values were obtained. Statistical analysis Homogeneity of variances and normal distribution were tested with Shapiro–Wilk test. Mean standard deviation and median (interquartile range defined as 25th–75th percentiles) were used to express normally and skewed continuous parameters, respectively. Frequencies and percentages were used to describe categorical parameters and were compared with chi-square test. Non-normally distributed parameters were compared with Bonferroni corrected Mann–Whitney U test and Wilcoxon test for paired samples. Spearman correlation analysis was performed to test the relation between non-normally distributed variables. Independent predictors for CAD were determined with binomial regression analysis. Analyses were performed using SPSS statistical software (version 21.0; SPSS Inc., Chicago, IL, USA). A two-tailed p < 0.05 was considered statistically significant.
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Results Macrophage infiltration and polarization in EAT Macrophage infiltration was increased in EAT of CAD patients. CAD patients had significantly higher CD68+ macrophage count in EAT when compared to the control group (p = 0.005). CD11c+ macrophage count was also significantly higher in patients with CAD when compared to that of the control group (p < 0.001). CD206+ macrophage count did not differ between groups. Although CD68+/total nuclei count ratio, which can be considered as a more objective parameter of macrophage infiltration, tended to be higher in EAT of the patient group, it did not reach statistical significance (p = 0.052). In the patient group, CD68+ macrophage infiltration was higher in the EAT to that of SAT (p = 0.001). CD68+/ total nuclei count ratio, CD11c+ and CD206+ macrophage counts were also significantly higher in the EAT of the patient group when compared to SAT of the patient group (p = 0.002, p = 0.003 and p = 0.002, respectively). None of the parameters related to the macrophage infiltration differed between EAT and SAT in the control group (Fig. 1). Macrophage polarization shifted into a pro-inflammatory profile in EAT of CAD patients. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1-macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the control group (p = 0.008). CD11c+/CD206+ macrophage ratio was also significantly higher in the EAT when compared to the
Fig. 1. Representative micrographs showing macrophages exhibiting CD68 immunoreactivity on (a) epicardial adipose tissue (EAT) of coronary artery disease (CAD) patients (n = 18), (b) subcutaneous adipose tissue (SAT) of CAD patients (n = 18), and (c) EAT of control group (n = 14). CD68-positive macrophages (asterisk) are mostly located at the perivascular region. Scale bar = 50 mm. CD68-positive cell count (d) and CD68/total nuclei count ratio (e) in EAT and SAT of patient and control groups are shown in the lower panel. # sign denotes statistical significance (p < 0.05).
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 2. (a) Representative micrographs showing CD11c (upper panel) and CD206 immune-positive cells (lower panel) in epicardial adipose tissue (EAT) of coronary artery disease (CAD) patients (n = 18), subcutaneous adipose tissue (SAT) of CAD patients (n = 18), and EAT of control group (n = 14). Scale bar = 50 mm. (b) CD11c+/CD206+ macrophage ratio in EAT and SAT of patient and control groups is shown. # sign denotes statistical significance (p < 0.05).
SAT of the patient group (p = 0.023). None of the macrophage polarization parameters differed between EAT and SAT in the control group (Fig. 2). Netrin-1, Unc5b, IL-12, and IL-18 expression in EAT and SAT Expression of netrin-1, Unc5b, and cytokines related to M1macrophage subtype (IL-12, IL-18) in EAT were increased in CAD patients. Patients with CAD had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p < 0.001, p < 0.001, and p = 0.006 respectively) (Fig. 3). None of the other parameters, including diabetes mellitus, hypertension, smoking, obesity [body mass index (BMI) 25 kg/ m2], or hyperlipidemia [low-density lipoprotein (LDL) 130 mg/ dL] were associated with the gene expression of netrin-1, Unc5b, IL12, and IL-18 in EAT (Table 2). Furthermore, no correlation was detected between netrin-1 gene expression and age, white blood cell count, and levels of LDL-cholesterol, high-density lipoprotein cholesterol or triglyceride. Netrin-1 expression was positively correlated with BMI (r = 0.287, p = 0.111) and Gensini score
(r = 0.387, p = 0.113); however, these did not reach statistical significance. Netrin-1 gene expression in EAT was positively correlated with gene expression of its receptor Unc5b in a statistically significant way (r = 0.590, p < 0.001). Netrin-1 gene expression was also positively and significantly correlated with IL-12 (r = 0.365, p = 0.040) and IL-18 (r = 0.386, p = 0.029) gene expression. Immunohistochemical analysis demonstrated netrin-1 and Unc5b staining on perivascular macrophages and endothelium in the adipose tissue. Both macrophages and endothelial cells exhibited moderate-to-high cytoplasmic immune reaction with netrin-1 and mild-to-moderate cytoplasmic immune reaction with Unc5b. Number of macrophages that were labeled with netrin-1 [15.00 (11.00–17.00) vs. 12.00 (10.75–13.00), p = 0.004] and Unc5b [9.00 (6.50–11.50) vs. 5.50 (5.00–6.50), p = 0.027] were significantly higher in EAT of the patient group when compared to that of the control group. Number of macrophages exhibiting netrin-1 (r = 0.609, p = 0.001) and Unc5b (r = 0.488, p = 0.010) immunoreactivity was correlated with their gene expression profile on qPCR in EAT (Fig. 4). Relationship between gene expression of netrin-1, Unc5b, IL-12, IL-18, and macrophage infiltration and polarization in EAT Netrin-1 and Unc5b expressions were found to be related with macrophage infiltration and polarization in EAT. Increased CD68+/ total nuclei count ratio (CD68+/total nuclei count ratio 0.11) was associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (Fig. 5a). Increased CD11c+/CD206+ macrophage ratio (CD11c+/CD206+ macrophage ratio 0.55) was found to be associated with higher gene expression of netrin-1, Unc5b, and IL-18 (Fig. 5b). Netrin-1 gene expression in EAT was also found to be positively correlated with CD68+/total nuclei count ratio in a statistically significant way (r = 0.649, p < 0.001). CD11c+/CD206+ macrophage count ratio was positively correlated (r = 0.369, p = 0.058) with netrin-1 gene expression in EAT; however, it did not reach statistical significance. Determination of predictors of CAD
Fig. 3. Netrin-1, Unc5b, IL-12, and IL-18 gene expression in epicardial adipose tissue of the patient (n = 18) and control (n = 14) groups. # sign denotes statistical significance (p < 0.05).
In the multivariate regression analysis model including LDLcholesterol and triglyceride levels, netrin-1 gene expression was found to be independently associated with presence of CAD (OR: 1.520, 95% CI: 1.123–2.058, p = 0.007).
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Table 2 Relationship between gene expression of netrin-1, Unc5b, IL-12, and IL-18, and baseline clinical and laboratory parameters. 2
DDCT
netrin-1
p-Value
DDCT
2
Unc5b
p-Value
2
DDCT
IL-12
p-Value
2
DDCT
IL-18
0.37 (0.13–1.20) <0.001* 0.69 (0.26–1.28) <0.001* 1.56 (0.65–2.14) 0.587 1.21 (0.43–1.85) CAD (n = 14) 2.98 (1.49–4.02) 1.48 (0.77–2.84) 2.77 (1.07–5.79) CAD+ 4.77 (2.13–16.01) (n = 18) 0.285 1.64 (0.82–3.15) 0.614 1.18 (0.66–2.08) 0.136 1.80 (0.90–4.04) DM 1.78 (0.28–6.21) (n = 27) 1.50 (0.25–6.89) 2.05 (1.62–2.87) 2.78 (0.44–5.12) 3.40 (1.80–12.99) DM+ (n = 5) 0.331 1.33 (0.52–3.31) 0.502 1.69 (0.72–2.59) 0.941 1.35 (0.77–2.78) 1.78 (0.37–4.77) HT (n = 17) 1.89 (0.99–3.05) 1.42 (0.75–2.06) 2.76 (1.02–4.94) 3.01 (0.88–14.27) HT+ (n = 15) 0.389 1.50 (0.91–2.79) 0.785 1.55 (0.86–2.82) 0.194 1.82 (0.84–4.76) Smoking 2.51 (0.67–10.29) (n = 21) 2.53 (0.36–3.61) 0.99 (0.54–1.95) 1.13 (0.75–2.82) Smoking+ 1.48 (0.28–3.32) (n = 11) 0.710 1.89 (0.77–3.47) 0.655 1.69 (0.66–2.08) 0.710 1.80 (1.06–4.94) 1.08 (0.24–14.27) LDL <130 mg/dL (n = 15) 1.45 (0.62–3.10) 1.18 (0.77–2.59) 1.82 (0.74–3.70) LDL 130 mg/dL 2.22 (1.48–3.91) (n = 17) 0.254 1.33 (0.52–2.72) 0.104 1.69 (0.86–2.22) 0.785 1.82 (0.96–3.70) BMI <25 kg/m2 1.87 (0.37–3.91) (n = 21) 2 2.67 (1.32–5.24) 1.41 (0.66–2.88) 1.63 (0.72–4.94) 3.32 (0.88–33.36) BMI 25 kg/m (n = 11) BMI, body mass index; CAD, coronary artery disease; DM, diabetes mellitus; HT, hypertension; IL, interleukin; LDL, low-density lipoprotein. * sign denotes statistical significance (p < 0.05).
Discussion This is the first study in the literature demonstrating enhanced netrin-1 and Unc5b expression in the EAT of patients with CAD, and the link between their expression and macrophage infiltration and polarization in EAT. Our study has shown that CD68+ macrophage infiltration was increased in EAT of patients with CAD when compared to the control group. This finding is compatible with the results of prior studies reporting inflammation in the EAT in patients with CAD, reflected by increased CD68+ macrophage infiltration [7,15]. On the contrary, Kitagawa et al. [16] have shown that, although more prominent in patients with CAD, CD68+ macrophage infiltration in EAT did not differ in a statistically significant way among patients who underwent coronary and non-coronary cardiac surgery. When macrophage subtypes are taken into consideration, our study has revealed that only CD11c+, but not CD206+, macrophage infiltration was significantly higher in EAT of the patient group when compared to the controls. On the other hand, the single previous study that evaluated macrophage subtypes in EAT in CAD, Hirata et al. [7] reported statistically significantly increased levels of both CD11c+ and CD206+ macrophages when compared to the control group. Increase in macrophage infiltration in the EAT of CAD patients was also confirmed in our study by comparing CD68+ macrophage infiltration in EAT and SAT of the patient group. Subjects with CAD had significantly higher CD68+ macrophage count and CD68+ macrophage/total nuclei count ratio in the EAT when compared to SAT. Similar findings were obtained in prior studies [7,16]. Our study also revealed that CD11c+ and CD206+ macrophage counts were also significantly higher in EAT when compared to SAT in subjects with CAD, in concordance with findings reported by Hirata et al. [7]. Our study demonstrated a shift to pro-inflammatory M1macrophage phenotype in EAT of the patient group when compared to the control group, reflected by increased CD11c+/ CD68+ and CD11c+/CD206+, and decreased CD206+/CD68+ macrophage ratios. M1/M2-macrophage ratio was also found to be higher in the EAT when compared to SAT in the patient group. Hirata et al. [7] have reported similar findings, suggesting that
p-Value 0.006*
1.000
0.370
0.785
0.502
0.611
pro-inflammatory macrophages are more dominant in EAT when both compared to the control group’s and to the patients’ SAT. Findings of our study are unique to demonstrate higher netrin-1 and Unc5b gene expression levels in EAT of subjects with CAD evaluated for the first time in the literature. Gene expression of Unc5b in EAT, which is considered to be the receptor involved in netrin-1’s effects on cardiovascular system, was found to be correlated with netrin-1 gene expression. Furthermore, netrin-1 expression was found to be associated with CAD presence independent of other cardiovascular risk factors. Although positively correlated with Gensini score, which is used to determine the severity of CAD, the correlation between Gensini score and netrin-1 expression failed to reach statistical significance possibly due to the relatively small study population. Studies have shown a wide spectrum of effects of netrin-1 on various tissues, including lungs, kidneys, and endothelial cells [10,17–21]. A single study that evaluated netrin-1 expression in the adipose tissue focused on netrin-1 expression in the omental adipose tissue and reported that it was higher in obese mice and humans, but not in non-obese, and was associated with macrophage retention [22]. In that study, upregulation of netrin-1 synthesis in macrophages with palmitate or exposing adipose tissue-located macrophages of non-obese mice to netrin-1 were shown to inhibit both migration response to chemokine (C–C motif) ligand 19 (CCL19), and dendritic cell and macrophage migration to the peripheral lymph nodes [22]. Furthermore, the aforementioned impaired migration response to CCL19 has been shown to be reversed with Unc5b blockage [22]. These findings have suggested that synthesis of netrin-1 by adipose tissue-located macrophages may lead to accumulation of macrophages and eventually lead to chronic, metabolic dysfunction originating from the adipose tissue. In our study, we demonstrated a positive correlation between netrin-1 expression and BMI, but it failed to reach statistical significance. Further studies are required to elucidate whether netrin-1 expression in EAT of subjects with CAD is independent of peripheral obesity. The role of netrin-1 in atherosclerosis has been investigated in a few studies [23]. Although netrin-1 has been shown to function as a
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 4. (a) Representative micrographs showing netrin-1 and Unc5b-positive cells (denoted by asterisk). Scale bar = 50 mm. (b) Correlation graphs for netrin-1 and Unc5b gene expression and immunohistochemical labeling are shown. EAT, epicardial adipose tissue; SAT, subcutaneous adipose tissue.
‘‘barrier’’ in other tissues that prevent infiltration of leukocytes, studies in atherosclerotic plaque have demonstrated pro-atherosclerotic properties related to netrin-1 expression by the foam cells. Recombinant netrin-1 secreted from in vitro foam cells has been reported to inhibit the migration response of macrophages to CCL19 [24,25]. van Gils et al. [26] have shown that human and mice atherosclerotic plaque foam cells expressed netrin-1 and Unc5b and selective deletion of netrin-1 in bone marrow of Lldr knockout mice resulted in smaller atherosclerotic lesion and decreased inflammatory activity within the plaque [26]. These findings have promoted the hypothesis that netrin-1 may act as a player in progression of atherosclerotic plaque via inhibition of cell regression and promotion of smooth muscle cell accumulation. Recently, Ramkhelawon et al. [27] have demonstrated that netrin-1 and Unc5b were expressed in the hypoxic regions of the mouse and human atherosclerotic plaques. Authors have demonstrated that netrin-1 and Unc5b expressions were induced with
hypoxia-inducible factor 1-alpha (HIF-1a), as well as oxidized LDL or oxidative stress [27]. This expression pattern has been attributed to hypoxia leading to impaired macrophage regression and enhanced macrophage survival [27]. Nevertheless, apart from the effects of netrin-1 on macrophage retention and survival which may be involved in enhanced inflammation in the EAT that aggravates coronary atherosclerosis and cardiac ischemia, it is also likely that inflammatory pathways that are associated with cardiac ischemia may lead to inflammation of EAT via paracrine actions, per se. These inflammatory pathways may be linked with various endogenous ligands that are called ‘‘danger-associated molecular patterns’’, and are known to be released upon injury and modulate inflammation in cardiac ischemia [28]. Our study has shown for the first time that increased macrophage infiltration was associated and positively correlated with netrin-1 expression in EAT. Netrin-1 expression in EAT was also greater in patients with predominantly M1-macrophage
Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016
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Fig. 5. Association of netrin-1, Unc5b, and M1-macrophage-related cytokine expression with (a) CD68/total nuclei count and (b) CD11c+/CD206+ macrophage ratio. # sign denotes statistical significance (p < 0.05).
phenotype. M1-macrophage-related cytokine IL-18 gene expression was found to be significantly higher in the EAT of the patient group when compared to the control group, which was associated with the predominancy of M1-macrophages. Although a similar association was not observed with IL-12 gene expression, netrin-1 expression was found to be correlated with both IL-12 and IL-18, suggesting another pro-inflammatory effect of netrin-1 in the EAT. To date, investigations to intervene the inflammation in the visceral adipose tissue have primarily focused on insulin sensitization. Nevertheless, these approaches fail since they do not target the initiating factors. We have demonstrated the involvement of netrin-1-Unc5b signaling pathway in EAT-originating or EATaccompanying chronic inflammation in coronary atherosclerosis for the first time. Inhibition of the netrin-1-Unc5b pathway may attenuate the inflammation in the EAT and the coronary arteries. Thus, molecular therapy targeting this pathway in EAT may be beneficial in preventing coronary atherosclerosis or its progression. Study limitations Our study has some limitations. First, the sample size is relatively small. However, this may not be considered as a major limitation, since previous studies with homologous study design also had similar size of study population and our findings reached statistical significance. Second, our study reveals an association, but not a clear causal relationship. Knockout animal model and in vitro cell culture studies may be implemented to explain the causality. Conclusions In conclusion, our study has shown an increase in netrin-1 expression that could be linked with macrophage infiltration and polarization in the EAT of patients with CAD. Demonstration of the involvement of this signaling pathway in EAT-originating chronic inflammation leading to atherosclerosis may have therapeutic implications in the future. Funding source This work was funded by Hacettepe University Scientific Research Projects Coordination Unit (Project number: 014 D11 101 003-705).
Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment Information reported in the article was presented at EAS Congress 2016.
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Please cite this article in press as: Gurses KM, et al. Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease. J Cardiol (2016), http://dx.doi.org/10.1016/j.jjcc.2016.08.016