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Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

Association of chemerin mRNA expression in human epicardial adipose tissue with coronary... Background: Growing evidence suggests that epicardial adipose tissue (EAT) may play a key role in the pathogenesis and development of coronary artery disease (CAD) by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Methods: Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous) were obtained from CAD (n = 37) and NCAD (n = 16) patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. Results: We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT), whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P < 0.05), moreover, this correlation remained statistically significant (r = 0.357, P < 0.05) after adjusting for age, gender, BMI and waist circumference. Chemerin mRNA expression in EAT was also positively correlated with BMI (r = 0.305, P < 0.05), waist circumference (r = 0.384, P < 0.01), fasting blood glucose (r = 0.334, P < 0.05) and negatively correlated with adiponectin mRNA expression in EAT (r = -0.322, P < 0.05). However, there were no significant differences in the serum levels of chemerin or adiponectin between the two groups. Likewise, neither serum chemerin nor serum adiponectin was associated with Gensini score (P > 0.05). Conclusions: The expressions of chemerin mRNA and protein are significantly higher in EAT from patients with CAD in Han Chinese patients. Furthermore, the severity of coronary atherosclerosis is positive correlated with the level of chemerin mRNA in EAT rather than its circulating level. Keywords: Epicardial adipose tissue, Chemerin, Adipokine, Atherosclerosis * Correspondence: [email protected] Department of Endocrinology, The Key Laboratory of Remodeling-related Cardiovascular Diseases, Beijing Anzhen Hospital, Capital Medical University, Ministry of Education, Beijing, 100029, China Full list of author information is available at the end of the article © 2011 Gao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 2 of 9 http://www.cardiab.com/content/10/1/87 surgery were enrolled in the study. Exclusion criteria Background included non ischemic cardiomyopathy, liver or renal Currently, obesity and accumulation of visceral adipose failure, neoplastic disease, thyroid or adrenal gland dis- tissue have been considered as the important risk factors ease, acute infectious disease, the aged (> 80 years) or for metabolic syndrome and coronary artery disease (CAD) patients taking corticosteroids, oral contraceptives, or [1]. Adipose tissue is now recognized as an active endo- psychotropic drugs. Patients were divided into CAD crine organ expressing and secreting several inflammatory group and NCAD group according to the results of mediators and cytokines, also known as adipokines, which selective coronary angiography. CAD was defined as the participate in the development of atherosclerosis [2]. Epicardial adipose tissue (EAT) is a type of visceral fat [3]. presence of stenoses of greater than 50% of the luminal diameter for at least one of the three major coronary Most recently, particular research interests have focused arteries. All CAD patients underwent elective coronary on EAT due to its close proximity to coronary arteries and artery bypass graft (CABG) surgery. All NCAD patients the plausibility that the adipokines derived from it may received elective open-heart surgery for valvular replace- have direct effects on each layer of the coronary wall via ment or atrial septal defect closure. These patients had paracrine and vasocrine pathways [4]. Previous studies no clinical signs of CAD and did not show significant have revealed that atherosclerotic intimal lesions are not coronary stenoses (≥50%) in the pre-operative coronary seen in the coronary arterial segments absent of EAT or angiographic examination. covered by myocardium [5], suggesting EAT may play a This study was approved by the ethics committee of role in the development of coronary atherosclerosis. Subse- Beijing Anzhen Hospital of Capital Medical University. quent studies have demonstrated that significantly lower This study complied with the declaration of Helsinki level of adiponectin and significantly higher levels of with the written informed consent obtained from all inflammatory cytokines exist in the EAT of patients with subjects prior to enrolment. CAD [6,7]. Recently, some evidence also proves that CAD is associated with increased chemokines production by Clinical data collection EAT [8-11]. In addition, infiltration of macrophages are Clinical data were obtained upon admission to hospital increased [12] and inflammatory pathways mediated by before surgery. Demographic data, medical history, and nuclear factor-B and Janus kinase (JNK) are activated in medications being used before surgery were recorded. In EAT in the presence of CAD [13]. All these data above addition, height, body weight, waist circumference and suggest EAT, due to its regional pro-inflammatory proper- blood pressure were measured. Then body mass index ties, may play an important role in the pathogenesis of CAD. (BMI) was calculated as weight (kg) divided by square of Chemerin, also known as tazarotene-induced gene 2 height (m ). (TIG2) or retinoic acid receptor responder 2 Gensini score assessment (RARRES2), functions as a chemotactic protein that Angiographic analyses were carried out by two experi- binds to the G protein-coupled receptor, CMKLR1 enced interventional cardiologists who were blinded to (chemR23), in humans [14]. Recently, it has been identi- the study protocol according to the result of coronary fied as a novel adipokine owing to its high expression angiography within six months prior to surgery. Then level in white adipocytes. Chemerin has been reported Gensini score was calculated to assess the severity of to be involved in modulating immune responses and coronary atherosclerosis according to the method glucolipid metabolism. Furthermore, some studies have described in the literature [16]. shown the strong association of serum chemerin level with markers of inflammation and components of the Blood samples detection metabolic syndrome [15]. Therefore, the double role of Peripheral venous blood samples were collected after over- chemerin in inflammation and metabolism may provide night fasting on the day after admission, and serum glu- an interesting link between obesity, inflammation and cose, lipid profiles, high sensitivity C-reactive protein atherosclerosis. However, the association of chemerin (hsCRP), insulin and hemoglobin A C (HbA C) were ana- with atherosclerosis has not yet been proved. In this 1 1 lyzed in central laboratory of Beijing Anzhen Hospital. study, we sought to determine the relationship of che- Insulin sensitivity was estimated by homeostasis model merin expression in human EAT and the severity of cor- assessment of insulin resistance (HOMA-IR) calculated as onary atherosclerosis. fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5. Methods Central venous blood was drawn before cardiopulmon- Subjects ary bypass during the operation. Then serum was stored Between October 2009 and March 2010, a total of 53 in aliquots at -80°C until being analyzed. Serum levels Han Chinese patients who underwent elective cardiac of chemerin and adiponectin were determined by Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 3 of 9 http://www.cardiab.com/content/10/1/87 commercially available enzyme-linked immunosorbent 25 μL containing 12.5 μL2×PCR-Mix,1 μL10 μmol/L assay (ELISA) kits (Adipobiotech, Beijing, China). each primer, and sterile water. The primers were designed using the Primer 5.0 software program, with their sites Adipose tissue acquisition spanning introns, so quantitation of the reaction was not Adipose biopsy samples were obtained prior to the initia- affected by the presence of genomic DNA. The primers tion of cardiopulmonary bypass from areas that had not used for amplification were shown in Table 1. Apart from previously been injured mechanically or cauterized. EAT the optimal annealing temperature and number of cycles biopsies (average 0.5 g) were taken near the proximal right of amplification, other conditions were the same in all coronary artery, and subcutaneous adipose tissue (SAT) PCR reactions. PCR was performed under the following samples were collected from the site of the chest incision. conditions: 3 minutes for an initial denaturation at 94°C, The specimens were rinsed with normal saline and divided followed by different cycles (30 s for denaturation at 94°C, into two portions. After removal of visible blood vessels, 30 s for annealing and 50 s for extension at 72°C), and 5 one portion was frozen immediately in liquid nitrogen and minutes for a final extension at 72°C. The number of PCR stored at -80°C for RNA isolation, another was immersed cycles in each system was chosen within linear phase in in neutralized formalin for immunohistochemical analysis. our preliminary trial to ensure the accuracy of semiquanti- tative analysis. A 5 μl aliquot of PCR products was then Immunohistochemistry analyzed using 1.5% agarose gel electrophoresis. Band Paraffin-embedded tissue sections were deparaffined and intensity for the target gene was quantified by densitome- rehydrated in descending grades of alcohol and stained try using Scion Image software and normalized to b-actin with hematoxylin and eosin. Selected serial sections were mRNA level to determine relative mRNA expression of subjected to immunohistochemistry with the Histostain- the target gene. Plus Kit LAB-SA Detection System (Invitrogen, USA) according to the manufacturer’s protocol. Briefly, sections Statistical analysis were incubated in 3% H O for 20 minutes followed by Variables such as serum adiponectin, hsCRP and trigly- 2 2 blocking with normal goat serum. After they were washed ceride with skewed distribution were log transformed. in PBS, sections were incubated with primary antibodies Continuous variables with normal distribution were (Chemerin, 1:200, Adipobiotech, Beijing, China) overnight expressed as mean ± SD, means were compared by paired at 4°C in a moisture chamber. Afterward, the slides were or unpaired Student’s t-test, as appropriate. Whereas for incubated with biotinylated secondary antibodies for data with skewed distribution, Mann-Whitney U test was 10~15 minutes followed by avidin-biotin for 15 minutes. used. Categorical variables were presented as percentages Sections were then exposed to DAB and counterstained and were analyzed by Chi-squared test. Associations with hematoxylin. Negative controls were carried out between the levels of adipokines in EAT and serum and omitting the primary antibody. Brightfield images were clinical variables were determined with the Pearson or observed with a light microscope and digital images were Spearman correlation coefficients. Partial correlation ana- recorded. Positive staining for chemerin was brown. lysis was performed to evaluate adjusted association Expression of chemerin was quantified by calculating the between chemerin mRNA level in EAT and Gensini score. integrated optical density (IOD) of positive staining tissue All statistical analyses were performed using SPSS 16.0 via Image-pro plus software. The IOD of each tissue sec- software, the P-value < 0.05 was considered statistically tion was calculated from eight different 400 magnified significant. fields. Results RNA extraction and Reverse Transcription-Polymerase Patient characteristics Chain Reaction (RT-PCR) The baseline characteristics of the two groups were shown Total RNA was extracted from adipose tissue samples in Table 2. In the CAD group (n = 37), 30 were men, and using Trizol reagent (Invitrogen, USA). The concentration mean age was 59.1 ± 8.0 years. In the NCAD group (n = and purity of isolated RNA were assessed by measuring 16), 12 were men, and mean age was 54.3 ± 9.4 years. As the optical density at 260 nm (OD260) and 280 nm expected, the majority of patients undergoing CABG sur- (OD280), and the integrity of RNA was also determined gery had multi-vessel lesions, moreover, obesity and by visualisation of 18S and 28S ribosomal bands. 1 μgof hypertension were more prevalent in the CAD group than RNA from each sample was reverse transcribed using in the NCAD group. There were no significant difference oligo(dT) and M-MLV Reverse Transcriptase (Promega, in age, gender, diabetes, left ventricular ejection fraction, USA), according to the manufacturer’s instructions. An blood pressure, triglycerides, total cholesterol, LDL choles- aliquot of 2 μL of the resulting cDNA was used for semi- terol, HbA C, serum creatinine, urea nitrogen, uric acid, quantitative PCR. PCR was carried out in a volume of medications treatment such as angiotensin converting Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 4 of 9 http://www.cardiab.com/content/10/1/87 Table 1 Primers for RT-PCR reaction Gene title Accession number Primer sequence Cycle Ta (°C) Product (bp) Chemerin NM_002889.3 F: 5’-GAAGAAACCCGAGTGCAAAG-3’ 28 56 229 R: 5’-CTTGGAGAAGGCGAACTGTC-3’ ChemR23 NM_001142343.1 F: 5’-CTCCCAATCCATATCACCTA-3’ 32 58 543 R: 5’-GCAGAGGAAGAAGGTAATGA-3’ Adiponectin NM_001177800.1 F: 5’-CTCCTCCTCACTTCCATTCTG-3’ 35 58 310 R: 5’-TTTCACCGATGTCTCCCTTA-3’ TNF-a NM_000594.2 F: 5’-TTCTGCCTGCTGCACTTTGGA-3’ 40 60 592 R: 5’-GGCGTTTGGGAAGGTTGGATG-3’ b-actin NM_001101.3 F: 5’-AGGTCATCACCATTGGCAAT-3’ 26 58 357 R: 5’-ACTCGTCATACTCCTGCTTG-3’ Abbreviations: TNF-a, tumor necrosis factor-a; Ta, annealing temperature. enzyme inhibitors/angiotensin II type 1 receptor blockers difference between the two groups (chemerin 0.94 ± 0.17 (ACEIs/ARBs), oral hypoglyceimic agents, insulin and vs. 0.78 ± 0.21, P <0.01; TNF-a 0.36 ± 0.34 vs. 0.15 ± antibiotics between the two groups. 0.19, P < 0.01; adiponectin 0.71 ± 0.15 vs. 0.83 ± 0.12, P < However, patients in the CAD group presented higher 0.01; chemR23 0.83 ± 0.45 vs. 0.62 ± 0.45, P = 0.140). levels of fasting glucose, HOMA-IR, hsCRP, and lower HDL cholesterol level compared to those in the NCAD Chemerin mRNA expression between EAT and SAT group. As for current medications therapy, aspirin, As for the comparison of paired EAT and SAT, 3 SAT nitrates, beta-blockers, statins, and calcium channel block- samples of CAD group were excluded from the analysis ers were prescribed more often in the CAD patients than due to insufficient adipose tissue biopsy samples or being in the NCAD patients. damaged during the operation. As shown in Figure 3, in the CAD group, significantly higher level of chemerin Immunohistochemical analysis mRNA was observed in EAT than in paired SAT (0.94 ± We performed immunohistochemistry to illustrate che- 0.17 vs. 0.84 ± 0.28, P < 0.05), whereas chemerin mRNA merin expression in adipose tissue. EAT and SAT samples level did not seem to differ between the two adipose were collected randomly from the CAD group (n = 6) and depots in the NCAD group (0.78 ± 0.21 vs. 0.82 ± 0.37, the NCAD group (n = 6), respectively. Figure 1A showed P > 0.05). Moreover, chemerin mRNA expression in SAT the representative slides of EAT and SAT from one patient of CAD group was comparable with that in SAT of with CAD (Figure 1A-a and 1A-b) and one without CAD NCAD group (0.84 ± 0.28 vs. 0.82 ± 0.37, P > 0.05). (Figure 1A-c and 1A-d). Immunohistochemical staining revealed that chemerin was expressed in both EAT and Association of EAT chemerin mRNA expression with SAT of the two groups. Furthermore, as shown in Figure clinical parameters 1B, quantitative analysis of immunohistochemistry Chemerin mRNA expression in EAT was positively cor- revealed that the amounts of chemerin protein in EAT related with Gensini score (r = 0.365, P < 0.05). Further- were higher in the patients with CAD than those without more, this correlation revealed by partial correlation CAD (70128.28 ± 13068.83 vs. 52312.03 ± 9899.90, P < analysis remained statistically significant (r = 0.357, P < 0.05). For the CAD group, significantly higher level of che- 0.05) even after adjusting for age, gender, BMI and waist merin protein was found in EAT than in SAT (70128.28 ± circumference. Chemerin mRNA expression in EAT was 13068.83 vs. 42942.04 ± 15460.67, P < 0.01), whereas no also found to be positively correlated with BMI (r = significant difference was found between EAT and SAT in 0.305, P < 0.05), waist circumference (r = 0.384, P < the NCAD group (52312.03 ± 9899.90 vs. 50533.71 ± 0.01), fasting glucose (r = 0.334, P < 0.05), chemR23 17289.54, P = 0.871). mRNA expression in EAT (r = 0.349, P < 0.05) and nega- tively correlated with adiponectin mRNA expression in EAT adipokines mRNA expression between CAD group EAT (r = -0.322, P < 0.05). However, no association was and NCAD group observed between chemerin mRNA expression in EAT and TNF-a mRNA expression in EAT (P > 0.05). As illustrated in Figure 2, we found significantly higher levels of chemerin, TNF-a mRNA and significantly lower level of adiponectin mRNA in EAT of CAD group com- Serum adipokines analysis pared to that of NCAD group, whereas the mRNA The serum samples of 33 CAD patients and 11 NCAD expression of chemR23 in EAT did not show significant patients met the criteria for analysis. The two groups Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 5 of 9 http://www.cardiab.com/content/10/1/87 Table 2 Baseline characteristics of CAD and NCAD groups before surgery CAD (n = 37) NCAD (n = 16) P Age (years) 59.1 ± 8.0 54.3 ± 9.4 NS Male (%) 30 (81.1) 12 (75.0) NS Hypertension (%) 27 (73.0) 3 (18.8) < 0.001 T DM (%) 11 (29.7) 1 (6.2) NS BMI (kg/m ) 27.1 ± 3.4 24.3 ± 2.6 0.004 Waist circumference (cm) 94.7 ± 9.2 85.5 ± 9.8 0.002 LVEF (%) 56.2 ± 10.1 60.1 ± 8.1 NS Systolic blood pressure (mmHg) 129.3 ± 20.7 127.1 ± 19.5 NS Diastolic blood pressure (mmHg) 78.1 ± 12.4 79 ± 14.0 NS Aspirin (%) 16 (43.2) 0 (0) 0.005 Nitrates (%) 37 (100) 0 (0) < 0.001 ACEIs/ARBs (%) 22 (59.5) 9 (56.2) NS Statins (%) 25 (67.6) 0 (0) < 0.001 b-blockers (%) 35 (94.6) 3 (18.8) < 0.001 Calcium channel blockers (%) 13 (35.1) 0 (0) 0.017 Insulin (%) 5 (13.5) 1 (6.2) NS Oral hypoglyceimic agents (%) 9 (24.3) 1 (6.2) NS Diuretics (%) 2 (5.4) 16 (100) < 0.001 Antibiotics (%) 6 (16.2) 4 (25.0) NS Fasting glucose (mmol/L) 6.08 (5.25, 7.45) 5.19 (4.86, 5.51) 0.002 HbA C (%) 6.3 ± 0.8 6.0 ± 0.6 NS Fasting insulin (μU/mL) 4.67 (2.43, 45.75) 2.27 (1.77, 3.06) 0.031 HOMA-IR 1.43 (0.69, 1.32) 0.53 (0.44, 0.68) 0.013 Total cholesterol (mmol/L) 4.44 ± 1.00 4.83 ± 0.73 NS HDL-C (mmol/L) 1.07 ± 0.24 1.25 ± 0.22 0.014 LDL-C (mmol/L) 2.68 ± 0.81 2.89 ± 0.53 NS Triglycerides (mmol/L) 1.88 ± 1.09 1.77 ± 1.49 NS Urea nitrogen (mmol/L) 6.82 ± 2.58 7.67 ± 2.46 NS Serum creatinine (μmol/L) 89.9 ± 21.3 83.5 ± 20.1 NS Uric acid (μmol/L) 380.2 ± 111.1 393.3 ± 96.8 NS hsCRP (mg/L) 1.95 (0.98, 6.75) 1.07 (0.51, 1.66) 0.003 Data are presented as mean ± SD, median (lower quartile, upper quartile), or number (%), P value represents CAD group vs. NCAD group. Abbreviations: CAD, coronary artery disease; T DM, type 2 diabetes; BMI: body mass index; LVEF, left ventricular ejection fraction; ACEIs: angiotensin converting enzyme inhibitors; ARBs: angiotensin II type 1 receptor blockers; HbA C, hemoglobin A C; HOMA-IR: homeostasis model assessment of insulin resistance; HDL-C, high-density 1 1 lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; hsCRP, high sensitivity C-reactive protein; NS, non-significance. were also age and gender matched. There were no sig- Discussion nificant differences in the serum levels of chemerin or In the present study, we found that EAT of CAD group adiponectin between the two groups (chemerin 118.13 ± showed significantly higher levels of chemerin mRNA 32.02 ng/ml vs. 99.60 ± 28.66 ng/ml, P = 0.096; adipo- and protein when compared with that of NCAD patients nectin 4.90 ± 1.39 μg/ml vs. 5.87 ± 1.52 μg/ml, P = in Han Chinese patients. Moreover, chemerin mRNA 0.155). Correlation analysis revealed that serum che- expression in EAT was positively correlated with the merin concentration was positively correlated with BMI severity of coronary atherosclerosis. However, there was (r = 0.323, P < 0.05), waist circumference (r = 0.398, P < no significant difference in the serum level of chemerin 0.01), HOMA-IR (r = 0.299, P < 0.05) and hsCRP (r = between the two groups. Likewise, no statistically signifi- 0.340, P < 0.05). However, neither serum chemerin (r = cant association was found between serum chemerin 0.295, P = 0.120) nor serum adiponectin (r = -0.328, P = level and coronary atherosclerosis. Spiroglou et al. have 0.083) level was found to be associated with Gensini reported a positive correlation between coronary athero- score. Also, there was no statistically significant correla- sclerosis and chemerin expression in pericoronary adi- tion between serum chemerin level and its expression pose tissue of autopsy cases [17], but their research was level in EAT (P > 0.05). not a case-control study, and whether the expression of Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 6 of 9 http://www.cardiab.com/content/10/1/87 Figure 1 Immunohistochemical analysis for chemerin in EAT and SAT. (A): the representative slides of EAT and SAT are from a patient with CAD (a and b) and a patient without CAD (c and d) separately (magnified × 400). Negative control with omission of primary antibody (e). (B): the result of quantitative analysis of immunohistochemistry for chemerin in EAT and SAT of the two groups (CAD group, n = 6; NCAD group, n = 6). * indicates P < 0.05, EAT of CAD group vs. EAT of NCAD group. ** indicates P < 0.01 as determined by paired t-test, EAT of CAD group vs. SAT of CAD group. Abbreviations: EAT, epicardial adipose tissue; SAT, subcutaneous adipose tissue; IOD, integrated optical density. chemerin was affected by the diseases that subjects suf- supported by the fact that significantly higher chemerin fered or by death were uncertain. Our study excluded the secretion from adipose tissue [19] as well as its circulat- established confounding factors which might influence ing level in obese patients [20] and significantly decreased the expression of chemerin and demonstrated the corre- serum chemerin level with weight loss after bariatric sur- lation of chemerin expression in EAT and the coronary gery [21]. With regard to the close association of che- atherosclerosis in Han Chinese patients, which was con- merin and obesity, a plausible interpretation was that sistent with the results of the study by Spiroglou et al. chronic low-grade inflammation that occurred in obesity Another study found serum chemerin level was not asso- might promote chemerin production by adipocytes ciated with coronary atherosclerotic plaque morphology [22,23]. On the other hand, chemerin could also promote as determined by computed tomography-angiography in the differentiation of preadipocytes in vitro [24], thereby patients with stable chest pain [15], which was consistent might contribute to obesity. with our findings. Most recently, Becker and his co-work- Previous studies have reported that chemerin mRNA ers reported that long-term overexpression of chemerin expression in EAT was associated with the indexes of obe- did not significantly affect extent of atherosclerotic lesion sity, and obesity has been considered as one of the most area in vivo [18]. Taken together, all these studies sug- important risk factors for CAD. In order to exclude the gested that locally produced chemerin by EAT rather effects of these potential confounders on coronary athero- sclerosis, we performed partial correlation analysis and than its circulating level might affect the atherosclerotic observed the correlation between chemerin mRNA process. As expected, we observed that both chemerin mRNA expression in EAT and coronary atherosclerosis remained expression in EAT and its serum level were positively statistically significant after adjusting for age, gender, BMI correlated with indexes of obesity (BMI and waist cir- and waist circumference. The association between EAT- cumference). The present findings were to some extent derived chemerin and coronary atherosclerosis could be Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 7 of 9 http://www.cardiab.com/content/10/1/87 Figure 2 The mRNA expression levels of adipokines in EAT between the two groups. (A): the result of agarose gel electrophoresis of PCR products in EAT. (B): the result of relative mRNA levels of chemerin, chemR23, adiponectin and TNF-a in EAT of the two groups (CAD group, n = 37; NCAD group, n = 16). ** indicates P < 0.01, CAD group vs. NCAD group. Abbreviations: a.u., arbitrary units. explained from two aspects below. First, significantly We also concluded that EAT of CAD group showed increased levels of inflammatory cytokines (TNF-a and significantly lower expression of adiponectin and signifi- IL-1b) in circulating and coronary blood of patients with cantly higher expression of TNF-a when compared with CAD could up-regulate chemerin in adjacent EAT via that of NCAD patients. This finding was confirmed by previous studies [6,7], suggested the pro- and anti- “inside to outside” signaling [22,23]. On the other hand, as inflammatory unbalance in EAT of patients with CAD. in vitro studies indicated, chemerin could recruit and acti- vate macrophages [25], promote cholesterol uptake [26] In addition, we observed a negative correlation between and induce endothelial angiogenesis in vitro [27], so we chemerin mRNA expression in EAT and adiponectin supposed that in vivo chemerin secreted by EAT could mRNA expression in EAT, which was consistent with also be transported downstream to interact with each the correlation of their protein levels [17]. layer of the coronary wall through “outside to inside” sig- Just as Mazurek et al. [28] illustrated that local inflam- naling [28], thus participating in several stages of the cor- matory burden in EAT did not correlate with their circu- onary atherosclerotic process: chemotaxis, foam cell lating concentrations, in the present study, no association formation and plaque destabilization. Hence we hypothe- was found between serum chemerin level and its expres- sized that EAT-derived chemerin and coronary athero- sion level in EAT. The following reasons could be put sclerosis might interact with each other. However, because forward to explain this phenomenon. First, we merely our research was a cross-sectional study, we could not detected chemerin at the transcription level which might draw any causal conclusions about their association. The not reflect the level of its protein translation and secre- direct effects of EAT-derived chemerin on coronary ather- tion. Second, EAT is only 1% of total fat mass, apart from osclerosis in vivo still needs to be further ascertained. EAT, other adipose depots as well as liver were also Previous studies have highlighted that significantly important sources of circulating chemerin level [24,30]. lower expression of adiponectin [7,29] and significantly Therefore, the contribution of EAT-derived chemerin to higher expression of chemokine (MCP-1) and several its systemic level might be negligible. inflammatory cytokines (TNF-a,IL-1b,IL-6) were Several limitations of the present study should be con- observed in EAT than in SAT [28]. In our study, we sidered. First, owing to the restriction of the amount of EAT biopsy samples, we merely detected chemerin also found chemerin mRNA expression was significantly mRNA and protein in EAT. It might have been useful to higher in EAT compared to paired SAT for CAD patients, whereas for NCAD patients, no significant dif- measure the level of chemerin secretion from EAT. Sec- ference was found between the two adipose depots. This ond, although the sample size of our study was relatively result indicated that EAT might play a more significant large in comparison with other similar studies on EAT, role in the pathogenesis of coronary atherosclerosis than we still could not carry out multivariate analysis to SAT. further clarify whether overexpression of chemerin in Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 8 of 9 http://www.cardiab.com/content/10/1/87 Figure 3 The mRNA expression level of chemerin between EAT and SAT. (A): the result of agarose gel electrophoresis of PCR products in EAT and SAT. (B): the scattered graph of relative mRNA levels of chemerin in EAT and SAT of the two groups (CAD group, n = 34; NCAD group, n = 16). EAT was an independent risk factor for coronary athero- aiming at exploring the direct effects of EAT-derived sclerosis. Finally, for serum analyses, the small serum chemerin on coronary atherosclerosis and its related sample size as well as the absence of serums of normal molecular mechanisms will provide a novel target for controls might have prevented us from achieving statisti- the prevention and treatment of CAD. cally significant associations. Acknowledgements Conclusions This study was supported by National Natural Science Foundation of China In conclusion, our study demonstrates that the expres- (No. 81100600) and the Funds of The Scientific Cooperation of Basic Medicine and Clinical Medicine of Capital Medical University (No.10JL54). We sions of chemerin mRNA and protein are significantly thank the cardiac surgeons Jinhua Li, Jingwei Li, Zhiqiang Luo, Liang Zhang, higher in EAT from patients with CAD in Han Chinese Ping Li, Lili Xu and Tao Bai at Beijing Anzhen Hospital for their assistance in patients. Furthermore, the severity of coronary athero- collecting adipose biopsy samples, and Qiang Shi for his assistance in revising the manuscript. Additionally, we also thank the patient volunteers sclerosis is associated with the level of chemerin mRNA for their participation. in EAT rather than its circulating level. Further studies Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 9 of 9 http://www.cardiab.com/content/10/1/87 Author details mediated inflammation in patients with coronary artery disease. J Clin Endocrinol Metab 2009, 94:261-267. Department of Endocrinology, The Key Laboratory of Remodeling-related 14. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, Cardiovascular Diseases, Beijing Anzhen Hospital, Capital Medical University, Brézillon S, Tyldesley R, Blanpain C, Detheux M, Mantovani A, Sozzani S, Ministry of Education, Beijing, 100029, China. Department of Special Care, Vassart G, Parmentier M, Communi D: Specific recruitment of antigen- Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China. presenting cells by chemerin, a novel processed ligand from human Department of Endocrinology, Peking Union Medical College Hospital, inflammatory fluids. J Exp Med 2003, 198:977-985. Peking Union Medical College, Chinese Academy of Medical Sciences, 15. Lehrke M, Becker A, Greif M, Stark R, Laubender RP, von Ziegler F, Beijing, 100730, China. Department of Cardiac Surgery, Beijing Anzhen Lebherz C, Tittus J, Reiser M, Becker C, Göke B, Leber AW, Parhofer KG, Hospital, Capital Medical University, Beijing, 100029, China. Broedl UC: Chemerin is associated with markers of inflammation and components of the metabolic syndrome but does not predict coronary Authors’ contributions XG carried out the molecular genetic studies and serum detection, atherosclerosis. Eur J Endocrinol 2009, 161:339-344. participated in the design of the study and drafted the manuscript. FZ 16. Gensini GG: A more meaningful scoring system for determining the carried out the immunoassays. XG, FZ and XZ involved in the acquisition of severity of coronary heart disease. Am J Cardiol 1983, 51:606. data as well as analysis and interpretation of data. YL and FG collected 17. Spiroglou SG, Kostopoulos CG, Varakis JN, Papadaki HH: Adipokines in adipose biopsy samples and serum samples. XG performed the statistical periaortic and epicardial adipose tissue: differential expression and analysis. HT and LW have been involved in revising the manuscript critically relation to atherosclerosis. J Atheroscler Thromb 2010, 17:115-130. for important intellectual content. HT, SM and FG conceived of the study, 18. Becker M, Rabe K, Lebherz C, Zugwurst J, Göke B, Parhofer KG, Lehrke M, and participated in its design and coordination. All authors have read and Broedl UC: Expression of human chemerin induces insulin resistance in approved the final manuscript. the skeletal muscle but does not affect weight, lipid levels, and atherosclerosis in LDL receptor knockout mice on high-fat diet. Diabetes Competing interests 2010, 59:2898-2903. The authors declare that they have no competing interests. 19. Sell H, Laurencikiene J, Taube A, Eckardt K, Cramer A, Horrighs A, Arner P, Eckel J: Chemerin is a novel adipocyte-derived factor inducing insulin Received: 16 July 2011 Accepted: 7 October 2011 resistance in primary human skeletal muscle cells. Diabetes 2009, Published: 7 October 2011 58:2731-2740. 20. Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, Walder K, Segal D: Chemerin is a novel adipokine associated with obesity and References metabolic syndrome. Endocrinology 2007, 148:4687-4694. 1. Mathieu P, Pibarot P, Larose E, Poirier P, Marette A, Després JP: Visceral 21. 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Horm Fasshauer M: Interleukin-1beta induces the novel adipokine chemerin in metab Res 2007, 39:707-709. adipocytes in vitro. Regul Pept 2009, 154:102-106. 5. Ishii T, Asuwa N, Masuda S, Ishikawa Y: The effects of a myocardial bridge 24. Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee SD, on coronary atherosclerosis and ischaemia. J Pathol 1998, 185:4-9. Muruganandan S, Sinal CJ: Chemerin, a novel adipokine that regulates 6. Eiras S, Teijeira-Fernández E, Shamagian LG, Fernandez AL, Vazquez- adipogenesis and adipocyte metabolism. J Biol Chem 2007, Boquete A, Gonzalez-Juanatey JR: Extension of coronary artery disease is 282:28175-28188. associated with increased IL-6 and decreased adiponectin gene 25. Wittamer V, Bondue B, Guillabert A, Vassart G, Parmentier M, Communi D: expression in epicardial adipose tissue. Cytokine 2008, 43:174-180. Neutrophil-mediated maturation of chemerin: a link between innate and 7. Zhou Y, Wei Y, Wang L, Wang X, Du X, Sun Z, Dong N, Chen X: Decreased adaptive immunity. J Immunol 2005, 175:487-493. adiponectin and increased inflammation expression in epicardial 26. McCarthy TC, Zuniga LA, Zabel BA, Butcher EC, Sinal CJ: The novel adipose tissue in coronary artery disease. Cardiovasc Diabetol 2011, 10:2. adipokine chemerin significantly increases cholesterol uptake in human 8. Henrichot E, Juge-Aubry CE, Pernin A, Pache JC, Velebit V, Dayer JM, macrophages [Abstract]. FASEB J 2008, 22:948, . Meda P, Chizzolini C, Meier CA: Production of chemokines by perivascular 27. Kaur J, Adya R, Tan BK, Chen J, Randeva HS: Identification of chemerin adipose tissue: a role in the pathogenesis of atherosclerosis? Arterioscler receptor (ChemR23) in human endothelial cells: Chemerin-induced Thromb Vasc Biol 2005, 25:2594-2599. endothelial angiogenesis. Biochem Biophys Res Commun 2010, 9. Shibasaki I, Nishikimi T, Mochizuki Y, Yamada Y, Yoshitatsu M, Inoue Y, 391:1762-1768. Kuwata T, Ogawa H, Tsuchiya G, Ishimitsu T, Fukuda H: Greater expression 28. Mazurek T, Zhang L, Zalewski A, Mannion JD, Diehl JT, Arafat H, Sarov- of inflammatory cytokines, adrenomedullin, and natriuretic peptide Blat L, O’Brien S, Keiper EA, Johnson AG, Martin J, Goldstein BJ, Shi Y: receptor-C in epicardial adipose tissue in coronary artery disease. Regul Human epicardial adipose tissue is a source of inflammatory mediators. Pept 2010, 165:210-217. Circulation 2003, 108:2460-2466. 10. Karastergiou K, Evans I, Ogston N, Miheisi N, Nair D, Kaski JC, Jahangiri M, 29. Bambace C, Telesca M, Zoico E, Sepe A, Olioso D, Rossi A, Corzato F, Di Mohamed-Ali V: Epicardial adipokines in obesity and coronary artery Francesco V, Mazzucco A, Santini F, Zamboni M: Adiponectin gene disease induce atherogenic changes in monocytes and endothelial cells. expression and adipocyte diameter: a comparison between epicardial Arterioscler Thromb Vasc Biol 2010, 30:1340-1346. and subcutaneous adipose tissue in men. Cardiovasc Pathol 2011, 20: 11. Madani R, Karastergiou K, Ogston NC, Miheisi N, Bhome R, Haloob N, e153-e156. Tan GD, Karpe F, Malone-Lee J, Hashemi M, Jahangiri M, Mohamed-Ali V: 30. Roh SG, Song SH, Choi KC, Katoh K, Wittamer V, Parmentier M, Sasaki S: RANTES release by human adipose tissue in vivo and evidence for Chemerin-a new adipokine that modulates adipogenesis via its own depot-specific differences. Am J Physiol Endocrinol Metab 2009, 296: receptor. Biochem Biophys Res Commun 2007, 362:1013-1018. E1262-E1268. 12. Baker AR, Silva NF, Quinn DW, Harte AL, Pagano D, Bonser RS, Kumar S, doi:10.1186/1475-2840-10-87 McTernan PG: Human epicardial adipose tissue expresses a pathogenic Cite this article as: Gao et al.: Association of chemerin mRNA expression profile of adipocytokines in patients with cardiovascular disease. in human epicardial adipose tissue with coronary atherosclerosis. Cardiovasc Diabetol 2006, 5:1. Cardiovascular Diabetology 2011 10:87. 13. Baker AR, Harte AL, Howell N, Pritlove DC, Ranasinghe AM, da Silva NF, Youssef EM, Khunti K, Davies MJ, Bonser RS, Kumar S, Pagano D, McTernan PG: Epicardial adipose tissue as a source of NFκB and JNK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cardiovascular Diabetology Springer Journals

Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

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Copyright © 2011 by Gao et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Diabetes; Angiology; Cardiology
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21981776
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Abstract

Background: Growing evidence suggests that epicardial adipose tissue (EAT) may play a key role in the pathogenesis and development of coronary artery disease (CAD) by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Methods: Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous) were obtained from CAD (n = 37) and NCAD (n = 16) patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. Results: We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT), whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P < 0.05), moreover, this correlation remained statistically significant (r = 0.357, P < 0.05) after adjusting for age, gender, BMI and waist circumference. Chemerin mRNA expression in EAT was also positively correlated with BMI (r = 0.305, P < 0.05), waist circumference (r = 0.384, P < 0.01), fasting blood glucose (r = 0.334, P < 0.05) and negatively correlated with adiponectin mRNA expression in EAT (r = -0.322, P < 0.05). However, there were no significant differences in the serum levels of chemerin or adiponectin between the two groups. Likewise, neither serum chemerin nor serum adiponectin was associated with Gensini score (P > 0.05). Conclusions: The expressions of chemerin mRNA and protein are significantly higher in EAT from patients with CAD in Han Chinese patients. Furthermore, the severity of coronary atherosclerosis is positive correlated with the level of chemerin mRNA in EAT rather than its circulating level. Keywords: Epicardial adipose tissue, Chemerin, Adipokine, Atherosclerosis * Correspondence: [email protected] Department of Endocrinology, The Key Laboratory of Remodeling-related Cardiovascular Diseases, Beijing Anzhen Hospital, Capital Medical University, Ministry of Education, Beijing, 100029, China Full list of author information is available at the end of the article © 2011 Gao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 2 of 9 http://www.cardiab.com/content/10/1/87 surgery were enrolled in the study. Exclusion criteria Background included non ischemic cardiomyopathy, liver or renal Currently, obesity and accumulation of visceral adipose failure, neoplastic disease, thyroid or adrenal gland dis- tissue have been considered as the important risk factors ease, acute infectious disease, the aged (> 80 years) or for metabolic syndrome and coronary artery disease (CAD) patients taking corticosteroids, oral contraceptives, or [1]. Adipose tissue is now recognized as an active endo- psychotropic drugs. Patients were divided into CAD crine organ expressing and secreting several inflammatory group and NCAD group according to the results of mediators and cytokines, also known as adipokines, which selective coronary angiography. CAD was defined as the participate in the development of atherosclerosis [2]. Epicardial adipose tissue (EAT) is a type of visceral fat [3]. presence of stenoses of greater than 50% of the luminal diameter for at least one of the three major coronary Most recently, particular research interests have focused arteries. All CAD patients underwent elective coronary on EAT due to its close proximity to coronary arteries and artery bypass graft (CABG) surgery. All NCAD patients the plausibility that the adipokines derived from it may received elective open-heart surgery for valvular replace- have direct effects on each layer of the coronary wall via ment or atrial septal defect closure. These patients had paracrine and vasocrine pathways [4]. Previous studies no clinical signs of CAD and did not show significant have revealed that atherosclerotic intimal lesions are not coronary stenoses (≥50%) in the pre-operative coronary seen in the coronary arterial segments absent of EAT or angiographic examination. covered by myocardium [5], suggesting EAT may play a This study was approved by the ethics committee of role in the development of coronary atherosclerosis. Subse- Beijing Anzhen Hospital of Capital Medical University. quent studies have demonstrated that significantly lower This study complied with the declaration of Helsinki level of adiponectin and significantly higher levels of with the written informed consent obtained from all inflammatory cytokines exist in the EAT of patients with subjects prior to enrolment. CAD [6,7]. Recently, some evidence also proves that CAD is associated with increased chemokines production by Clinical data collection EAT [8-11]. In addition, infiltration of macrophages are Clinical data were obtained upon admission to hospital increased [12] and inflammatory pathways mediated by before surgery. Demographic data, medical history, and nuclear factor-B and Janus kinase (JNK) are activated in medications being used before surgery were recorded. In EAT in the presence of CAD [13]. All these data above addition, height, body weight, waist circumference and suggest EAT, due to its regional pro-inflammatory proper- blood pressure were measured. Then body mass index ties, may play an important role in the pathogenesis of CAD. (BMI) was calculated as weight (kg) divided by square of Chemerin, also known as tazarotene-induced gene 2 height (m ). (TIG2) or retinoic acid receptor responder 2 Gensini score assessment (RARRES2), functions as a chemotactic protein that Angiographic analyses were carried out by two experi- binds to the G protein-coupled receptor, CMKLR1 enced interventional cardiologists who were blinded to (chemR23), in humans [14]. Recently, it has been identi- the study protocol according to the result of coronary fied as a novel adipokine owing to its high expression angiography within six months prior to surgery. Then level in white adipocytes. Chemerin has been reported Gensini score was calculated to assess the severity of to be involved in modulating immune responses and coronary atherosclerosis according to the method glucolipid metabolism. Furthermore, some studies have described in the literature [16]. shown the strong association of serum chemerin level with markers of inflammation and components of the Blood samples detection metabolic syndrome [15]. Therefore, the double role of Peripheral venous blood samples were collected after over- chemerin in inflammation and metabolism may provide night fasting on the day after admission, and serum glu- an interesting link between obesity, inflammation and cose, lipid profiles, high sensitivity C-reactive protein atherosclerosis. However, the association of chemerin (hsCRP), insulin and hemoglobin A C (HbA C) were ana- with atherosclerosis has not yet been proved. In this 1 1 lyzed in central laboratory of Beijing Anzhen Hospital. study, we sought to determine the relationship of che- Insulin sensitivity was estimated by homeostasis model merin expression in human EAT and the severity of cor- assessment of insulin resistance (HOMA-IR) calculated as onary atherosclerosis. fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5. Methods Central venous blood was drawn before cardiopulmon- Subjects ary bypass during the operation. Then serum was stored Between October 2009 and March 2010, a total of 53 in aliquots at -80°C until being analyzed. Serum levels Han Chinese patients who underwent elective cardiac of chemerin and adiponectin were determined by Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 3 of 9 http://www.cardiab.com/content/10/1/87 commercially available enzyme-linked immunosorbent 25 μL containing 12.5 μL2×PCR-Mix,1 μL10 μmol/L assay (ELISA) kits (Adipobiotech, Beijing, China). each primer, and sterile water. The primers were designed using the Primer 5.0 software program, with their sites Adipose tissue acquisition spanning introns, so quantitation of the reaction was not Adipose biopsy samples were obtained prior to the initia- affected by the presence of genomic DNA. The primers tion of cardiopulmonary bypass from areas that had not used for amplification were shown in Table 1. Apart from previously been injured mechanically or cauterized. EAT the optimal annealing temperature and number of cycles biopsies (average 0.5 g) were taken near the proximal right of amplification, other conditions were the same in all coronary artery, and subcutaneous adipose tissue (SAT) PCR reactions. PCR was performed under the following samples were collected from the site of the chest incision. conditions: 3 minutes for an initial denaturation at 94°C, The specimens were rinsed with normal saline and divided followed by different cycles (30 s for denaturation at 94°C, into two portions. After removal of visible blood vessels, 30 s for annealing and 50 s for extension at 72°C), and 5 one portion was frozen immediately in liquid nitrogen and minutes for a final extension at 72°C. The number of PCR stored at -80°C for RNA isolation, another was immersed cycles in each system was chosen within linear phase in in neutralized formalin for immunohistochemical analysis. our preliminary trial to ensure the accuracy of semiquanti- tative analysis. A 5 μl aliquot of PCR products was then Immunohistochemistry analyzed using 1.5% agarose gel electrophoresis. Band Paraffin-embedded tissue sections were deparaffined and intensity for the target gene was quantified by densitome- rehydrated in descending grades of alcohol and stained try using Scion Image software and normalized to b-actin with hematoxylin and eosin. Selected serial sections were mRNA level to determine relative mRNA expression of subjected to immunohistochemistry with the Histostain- the target gene. Plus Kit LAB-SA Detection System (Invitrogen, USA) according to the manufacturer’s protocol. Briefly, sections Statistical analysis were incubated in 3% H O for 20 minutes followed by Variables such as serum adiponectin, hsCRP and trigly- 2 2 blocking with normal goat serum. After they were washed ceride with skewed distribution were log transformed. in PBS, sections were incubated with primary antibodies Continuous variables with normal distribution were (Chemerin, 1:200, Adipobiotech, Beijing, China) overnight expressed as mean ± SD, means were compared by paired at 4°C in a moisture chamber. Afterward, the slides were or unpaired Student’s t-test, as appropriate. Whereas for incubated with biotinylated secondary antibodies for data with skewed distribution, Mann-Whitney U test was 10~15 minutes followed by avidin-biotin for 15 minutes. used. Categorical variables were presented as percentages Sections were then exposed to DAB and counterstained and were analyzed by Chi-squared test. Associations with hematoxylin. Negative controls were carried out between the levels of adipokines in EAT and serum and omitting the primary antibody. Brightfield images were clinical variables were determined with the Pearson or observed with a light microscope and digital images were Spearman correlation coefficients. Partial correlation ana- recorded. Positive staining for chemerin was brown. lysis was performed to evaluate adjusted association Expression of chemerin was quantified by calculating the between chemerin mRNA level in EAT and Gensini score. integrated optical density (IOD) of positive staining tissue All statistical analyses were performed using SPSS 16.0 via Image-pro plus software. The IOD of each tissue sec- software, the P-value < 0.05 was considered statistically tion was calculated from eight different 400 magnified significant. fields. Results RNA extraction and Reverse Transcription-Polymerase Patient characteristics Chain Reaction (RT-PCR) The baseline characteristics of the two groups were shown Total RNA was extracted from adipose tissue samples in Table 2. In the CAD group (n = 37), 30 were men, and using Trizol reagent (Invitrogen, USA). The concentration mean age was 59.1 ± 8.0 years. In the NCAD group (n = and purity of isolated RNA were assessed by measuring 16), 12 were men, and mean age was 54.3 ± 9.4 years. As the optical density at 260 nm (OD260) and 280 nm expected, the majority of patients undergoing CABG sur- (OD280), and the integrity of RNA was also determined gery had multi-vessel lesions, moreover, obesity and by visualisation of 18S and 28S ribosomal bands. 1 μgof hypertension were more prevalent in the CAD group than RNA from each sample was reverse transcribed using in the NCAD group. There were no significant difference oligo(dT) and M-MLV Reverse Transcriptase (Promega, in age, gender, diabetes, left ventricular ejection fraction, USA), according to the manufacturer’s instructions. An blood pressure, triglycerides, total cholesterol, LDL choles- aliquot of 2 μL of the resulting cDNA was used for semi- terol, HbA C, serum creatinine, urea nitrogen, uric acid, quantitative PCR. PCR was carried out in a volume of medications treatment such as angiotensin converting Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 4 of 9 http://www.cardiab.com/content/10/1/87 Table 1 Primers for RT-PCR reaction Gene title Accession number Primer sequence Cycle Ta (°C) Product (bp) Chemerin NM_002889.3 F: 5’-GAAGAAACCCGAGTGCAAAG-3’ 28 56 229 R: 5’-CTTGGAGAAGGCGAACTGTC-3’ ChemR23 NM_001142343.1 F: 5’-CTCCCAATCCATATCACCTA-3’ 32 58 543 R: 5’-GCAGAGGAAGAAGGTAATGA-3’ Adiponectin NM_001177800.1 F: 5’-CTCCTCCTCACTTCCATTCTG-3’ 35 58 310 R: 5’-TTTCACCGATGTCTCCCTTA-3’ TNF-a NM_000594.2 F: 5’-TTCTGCCTGCTGCACTTTGGA-3’ 40 60 592 R: 5’-GGCGTTTGGGAAGGTTGGATG-3’ b-actin NM_001101.3 F: 5’-AGGTCATCACCATTGGCAAT-3’ 26 58 357 R: 5’-ACTCGTCATACTCCTGCTTG-3’ Abbreviations: TNF-a, tumor necrosis factor-a; Ta, annealing temperature. enzyme inhibitors/angiotensin II type 1 receptor blockers difference between the two groups (chemerin 0.94 ± 0.17 (ACEIs/ARBs), oral hypoglyceimic agents, insulin and vs. 0.78 ± 0.21, P <0.01; TNF-a 0.36 ± 0.34 vs. 0.15 ± antibiotics between the two groups. 0.19, P < 0.01; adiponectin 0.71 ± 0.15 vs. 0.83 ± 0.12, P < However, patients in the CAD group presented higher 0.01; chemR23 0.83 ± 0.45 vs. 0.62 ± 0.45, P = 0.140). levels of fasting glucose, HOMA-IR, hsCRP, and lower HDL cholesterol level compared to those in the NCAD Chemerin mRNA expression between EAT and SAT group. As for current medications therapy, aspirin, As for the comparison of paired EAT and SAT, 3 SAT nitrates, beta-blockers, statins, and calcium channel block- samples of CAD group were excluded from the analysis ers were prescribed more often in the CAD patients than due to insufficient adipose tissue biopsy samples or being in the NCAD patients. damaged during the operation. As shown in Figure 3, in the CAD group, significantly higher level of chemerin Immunohistochemical analysis mRNA was observed in EAT than in paired SAT (0.94 ± We performed immunohistochemistry to illustrate che- 0.17 vs. 0.84 ± 0.28, P < 0.05), whereas chemerin mRNA merin expression in adipose tissue. EAT and SAT samples level did not seem to differ between the two adipose were collected randomly from the CAD group (n = 6) and depots in the NCAD group (0.78 ± 0.21 vs. 0.82 ± 0.37, the NCAD group (n = 6), respectively. Figure 1A showed P > 0.05). Moreover, chemerin mRNA expression in SAT the representative slides of EAT and SAT from one patient of CAD group was comparable with that in SAT of with CAD (Figure 1A-a and 1A-b) and one without CAD NCAD group (0.84 ± 0.28 vs. 0.82 ± 0.37, P > 0.05). (Figure 1A-c and 1A-d). Immunohistochemical staining revealed that chemerin was expressed in both EAT and Association of EAT chemerin mRNA expression with SAT of the two groups. Furthermore, as shown in Figure clinical parameters 1B, quantitative analysis of immunohistochemistry Chemerin mRNA expression in EAT was positively cor- revealed that the amounts of chemerin protein in EAT related with Gensini score (r = 0.365, P < 0.05). Further- were higher in the patients with CAD than those without more, this correlation revealed by partial correlation CAD (70128.28 ± 13068.83 vs. 52312.03 ± 9899.90, P < analysis remained statistically significant (r = 0.357, P < 0.05). For the CAD group, significantly higher level of che- 0.05) even after adjusting for age, gender, BMI and waist merin protein was found in EAT than in SAT (70128.28 ± circumference. Chemerin mRNA expression in EAT was 13068.83 vs. 42942.04 ± 15460.67, P < 0.01), whereas no also found to be positively correlated with BMI (r = significant difference was found between EAT and SAT in 0.305, P < 0.05), waist circumference (r = 0.384, P < the NCAD group (52312.03 ± 9899.90 vs. 50533.71 ± 0.01), fasting glucose (r = 0.334, P < 0.05), chemR23 17289.54, P = 0.871). mRNA expression in EAT (r = 0.349, P < 0.05) and nega- tively correlated with adiponectin mRNA expression in EAT adipokines mRNA expression between CAD group EAT (r = -0.322, P < 0.05). However, no association was and NCAD group observed between chemerin mRNA expression in EAT and TNF-a mRNA expression in EAT (P > 0.05). As illustrated in Figure 2, we found significantly higher levels of chemerin, TNF-a mRNA and significantly lower level of adiponectin mRNA in EAT of CAD group com- Serum adipokines analysis pared to that of NCAD group, whereas the mRNA The serum samples of 33 CAD patients and 11 NCAD expression of chemR23 in EAT did not show significant patients met the criteria for analysis. The two groups Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 5 of 9 http://www.cardiab.com/content/10/1/87 Table 2 Baseline characteristics of CAD and NCAD groups before surgery CAD (n = 37) NCAD (n = 16) P Age (years) 59.1 ± 8.0 54.3 ± 9.4 NS Male (%) 30 (81.1) 12 (75.0) NS Hypertension (%) 27 (73.0) 3 (18.8) < 0.001 T DM (%) 11 (29.7) 1 (6.2) NS BMI (kg/m ) 27.1 ± 3.4 24.3 ± 2.6 0.004 Waist circumference (cm) 94.7 ± 9.2 85.5 ± 9.8 0.002 LVEF (%) 56.2 ± 10.1 60.1 ± 8.1 NS Systolic blood pressure (mmHg) 129.3 ± 20.7 127.1 ± 19.5 NS Diastolic blood pressure (mmHg) 78.1 ± 12.4 79 ± 14.0 NS Aspirin (%) 16 (43.2) 0 (0) 0.005 Nitrates (%) 37 (100) 0 (0) < 0.001 ACEIs/ARBs (%) 22 (59.5) 9 (56.2) NS Statins (%) 25 (67.6) 0 (0) < 0.001 b-blockers (%) 35 (94.6) 3 (18.8) < 0.001 Calcium channel blockers (%) 13 (35.1) 0 (0) 0.017 Insulin (%) 5 (13.5) 1 (6.2) NS Oral hypoglyceimic agents (%) 9 (24.3) 1 (6.2) NS Diuretics (%) 2 (5.4) 16 (100) < 0.001 Antibiotics (%) 6 (16.2) 4 (25.0) NS Fasting glucose (mmol/L) 6.08 (5.25, 7.45) 5.19 (4.86, 5.51) 0.002 HbA C (%) 6.3 ± 0.8 6.0 ± 0.6 NS Fasting insulin (μU/mL) 4.67 (2.43, 45.75) 2.27 (1.77, 3.06) 0.031 HOMA-IR 1.43 (0.69, 1.32) 0.53 (0.44, 0.68) 0.013 Total cholesterol (mmol/L) 4.44 ± 1.00 4.83 ± 0.73 NS HDL-C (mmol/L) 1.07 ± 0.24 1.25 ± 0.22 0.014 LDL-C (mmol/L) 2.68 ± 0.81 2.89 ± 0.53 NS Triglycerides (mmol/L) 1.88 ± 1.09 1.77 ± 1.49 NS Urea nitrogen (mmol/L) 6.82 ± 2.58 7.67 ± 2.46 NS Serum creatinine (μmol/L) 89.9 ± 21.3 83.5 ± 20.1 NS Uric acid (μmol/L) 380.2 ± 111.1 393.3 ± 96.8 NS hsCRP (mg/L) 1.95 (0.98, 6.75) 1.07 (0.51, 1.66) 0.003 Data are presented as mean ± SD, median (lower quartile, upper quartile), or number (%), P value represents CAD group vs. NCAD group. Abbreviations: CAD, coronary artery disease; T DM, type 2 diabetes; BMI: body mass index; LVEF, left ventricular ejection fraction; ACEIs: angiotensin converting enzyme inhibitors; ARBs: angiotensin II type 1 receptor blockers; HbA C, hemoglobin A C; HOMA-IR: homeostasis model assessment of insulin resistance; HDL-C, high-density 1 1 lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; hsCRP, high sensitivity C-reactive protein; NS, non-significance. were also age and gender matched. There were no sig- Discussion nificant differences in the serum levels of chemerin or In the present study, we found that EAT of CAD group adiponectin between the two groups (chemerin 118.13 ± showed significantly higher levels of chemerin mRNA 32.02 ng/ml vs. 99.60 ± 28.66 ng/ml, P = 0.096; adipo- and protein when compared with that of NCAD patients nectin 4.90 ± 1.39 μg/ml vs. 5.87 ± 1.52 μg/ml, P = in Han Chinese patients. Moreover, chemerin mRNA 0.155). Correlation analysis revealed that serum che- expression in EAT was positively correlated with the merin concentration was positively correlated with BMI severity of coronary atherosclerosis. However, there was (r = 0.323, P < 0.05), waist circumference (r = 0.398, P < no significant difference in the serum level of chemerin 0.01), HOMA-IR (r = 0.299, P < 0.05) and hsCRP (r = between the two groups. Likewise, no statistically signifi- 0.340, P < 0.05). However, neither serum chemerin (r = cant association was found between serum chemerin 0.295, P = 0.120) nor serum adiponectin (r = -0.328, P = level and coronary atherosclerosis. Spiroglou et al. have 0.083) level was found to be associated with Gensini reported a positive correlation between coronary athero- score. Also, there was no statistically significant correla- sclerosis and chemerin expression in pericoronary adi- tion between serum chemerin level and its expression pose tissue of autopsy cases [17], but their research was level in EAT (P > 0.05). not a case-control study, and whether the expression of Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 6 of 9 http://www.cardiab.com/content/10/1/87 Figure 1 Immunohistochemical analysis for chemerin in EAT and SAT. (A): the representative slides of EAT and SAT are from a patient with CAD (a and b) and a patient without CAD (c and d) separately (magnified × 400). Negative control with omission of primary antibody (e). (B): the result of quantitative analysis of immunohistochemistry for chemerin in EAT and SAT of the two groups (CAD group, n = 6; NCAD group, n = 6). * indicates P < 0.05, EAT of CAD group vs. EAT of NCAD group. ** indicates P < 0.01 as determined by paired t-test, EAT of CAD group vs. SAT of CAD group. Abbreviations: EAT, epicardial adipose tissue; SAT, subcutaneous adipose tissue; IOD, integrated optical density. chemerin was affected by the diseases that subjects suf- supported by the fact that significantly higher chemerin fered or by death were uncertain. Our study excluded the secretion from adipose tissue [19] as well as its circulat- established confounding factors which might influence ing level in obese patients [20] and significantly decreased the expression of chemerin and demonstrated the corre- serum chemerin level with weight loss after bariatric sur- lation of chemerin expression in EAT and the coronary gery [21]. With regard to the close association of che- atherosclerosis in Han Chinese patients, which was con- merin and obesity, a plausible interpretation was that sistent with the results of the study by Spiroglou et al. chronic low-grade inflammation that occurred in obesity Another study found serum chemerin level was not asso- might promote chemerin production by adipocytes ciated with coronary atherosclerotic plaque morphology [22,23]. On the other hand, chemerin could also promote as determined by computed tomography-angiography in the differentiation of preadipocytes in vitro [24], thereby patients with stable chest pain [15], which was consistent might contribute to obesity. with our findings. Most recently, Becker and his co-work- Previous studies have reported that chemerin mRNA ers reported that long-term overexpression of chemerin expression in EAT was associated with the indexes of obe- did not significantly affect extent of atherosclerotic lesion sity, and obesity has been considered as one of the most area in vivo [18]. Taken together, all these studies sug- important risk factors for CAD. In order to exclude the gested that locally produced chemerin by EAT rather effects of these potential confounders on coronary athero- sclerosis, we performed partial correlation analysis and than its circulating level might affect the atherosclerotic observed the correlation between chemerin mRNA process. As expected, we observed that both chemerin mRNA expression in EAT and coronary atherosclerosis remained expression in EAT and its serum level were positively statistically significant after adjusting for age, gender, BMI correlated with indexes of obesity (BMI and waist cir- and waist circumference. The association between EAT- cumference). The present findings were to some extent derived chemerin and coronary atherosclerosis could be Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 7 of 9 http://www.cardiab.com/content/10/1/87 Figure 2 The mRNA expression levels of adipokines in EAT between the two groups. (A): the result of agarose gel electrophoresis of PCR products in EAT. (B): the result of relative mRNA levels of chemerin, chemR23, adiponectin and TNF-a in EAT of the two groups (CAD group, n = 37; NCAD group, n = 16). ** indicates P < 0.01, CAD group vs. NCAD group. Abbreviations: a.u., arbitrary units. explained from two aspects below. First, significantly We also concluded that EAT of CAD group showed increased levels of inflammatory cytokines (TNF-a and significantly lower expression of adiponectin and signifi- IL-1b) in circulating and coronary blood of patients with cantly higher expression of TNF-a when compared with CAD could up-regulate chemerin in adjacent EAT via that of NCAD patients. This finding was confirmed by previous studies [6,7], suggested the pro- and anti- “inside to outside” signaling [22,23]. On the other hand, as inflammatory unbalance in EAT of patients with CAD. in vitro studies indicated, chemerin could recruit and acti- vate macrophages [25], promote cholesterol uptake [26] In addition, we observed a negative correlation between and induce endothelial angiogenesis in vitro [27], so we chemerin mRNA expression in EAT and adiponectin supposed that in vivo chemerin secreted by EAT could mRNA expression in EAT, which was consistent with also be transported downstream to interact with each the correlation of their protein levels [17]. layer of the coronary wall through “outside to inside” sig- Just as Mazurek et al. [28] illustrated that local inflam- naling [28], thus participating in several stages of the cor- matory burden in EAT did not correlate with their circu- onary atherosclerotic process: chemotaxis, foam cell lating concentrations, in the present study, no association formation and plaque destabilization. Hence we hypothe- was found between serum chemerin level and its expres- sized that EAT-derived chemerin and coronary athero- sion level in EAT. The following reasons could be put sclerosis might interact with each other. However, because forward to explain this phenomenon. First, we merely our research was a cross-sectional study, we could not detected chemerin at the transcription level which might draw any causal conclusions about their association. The not reflect the level of its protein translation and secre- direct effects of EAT-derived chemerin on coronary ather- tion. Second, EAT is only 1% of total fat mass, apart from osclerosis in vivo still needs to be further ascertained. EAT, other adipose depots as well as liver were also Previous studies have highlighted that significantly important sources of circulating chemerin level [24,30]. lower expression of adiponectin [7,29] and significantly Therefore, the contribution of EAT-derived chemerin to higher expression of chemokine (MCP-1) and several its systemic level might be negligible. inflammatory cytokines (TNF-a,IL-1b,IL-6) were Several limitations of the present study should be con- observed in EAT than in SAT [28]. In our study, we sidered. First, owing to the restriction of the amount of EAT biopsy samples, we merely detected chemerin also found chemerin mRNA expression was significantly mRNA and protein in EAT. It might have been useful to higher in EAT compared to paired SAT for CAD patients, whereas for NCAD patients, no significant dif- measure the level of chemerin secretion from EAT. Sec- ference was found between the two adipose depots. This ond, although the sample size of our study was relatively result indicated that EAT might play a more significant large in comparison with other similar studies on EAT, role in the pathogenesis of coronary atherosclerosis than we still could not carry out multivariate analysis to SAT. further clarify whether overexpression of chemerin in Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 8 of 9 http://www.cardiab.com/content/10/1/87 Figure 3 The mRNA expression level of chemerin between EAT and SAT. (A): the result of agarose gel electrophoresis of PCR products in EAT and SAT. (B): the scattered graph of relative mRNA levels of chemerin in EAT and SAT of the two groups (CAD group, n = 34; NCAD group, n = 16). EAT was an independent risk factor for coronary athero- aiming at exploring the direct effects of EAT-derived sclerosis. Finally, for serum analyses, the small serum chemerin on coronary atherosclerosis and its related sample size as well as the absence of serums of normal molecular mechanisms will provide a novel target for controls might have prevented us from achieving statisti- the prevention and treatment of CAD. cally significant associations. Acknowledgements Conclusions This study was supported by National Natural Science Foundation of China In conclusion, our study demonstrates that the expres- (No. 81100600) and the Funds of The Scientific Cooperation of Basic Medicine and Clinical Medicine of Capital Medical University (No.10JL54). We sions of chemerin mRNA and protein are significantly thank the cardiac surgeons Jinhua Li, Jingwei Li, Zhiqiang Luo, Liang Zhang, higher in EAT from patients with CAD in Han Chinese Ping Li, Lili Xu and Tao Bai at Beijing Anzhen Hospital for their assistance in patients. Furthermore, the severity of coronary athero- collecting adipose biopsy samples, and Qiang Shi for his assistance in revising the manuscript. Additionally, we also thank the patient volunteers sclerosis is associated with the level of chemerin mRNA for their participation. in EAT rather than its circulating level. Further studies Gao et al. Cardiovascular Diabetology 2011, 10:87 Page 9 of 9 http://www.cardiab.com/content/10/1/87 Author details mediated inflammation in patients with coronary artery disease. J Clin Endocrinol Metab 2009, 94:261-267. Department of Endocrinology, The Key Laboratory of Remodeling-related 14. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, Cardiovascular Diseases, Beijing Anzhen Hospital, Capital Medical University, Brézillon S, Tyldesley R, Blanpain C, Detheux M, Mantovani A, Sozzani S, Ministry of Education, Beijing, 100029, China. Department of Special Care, Vassart G, Parmentier M, Communi D: Specific recruitment of antigen- Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China. presenting cells by chemerin, a novel processed ligand from human Department of Endocrinology, Peking Union Medical College Hospital, inflammatory fluids. J Exp Med 2003, 198:977-985. Peking Union Medical College, Chinese Academy of Medical Sciences, 15. Lehrke M, Becker A, Greif M, Stark R, Laubender RP, von Ziegler F, Beijing, 100730, China. Department of Cardiac Surgery, Beijing Anzhen Lebherz C, Tittus J, Reiser M, Becker C, Göke B, Leber AW, Parhofer KG, Hospital, Capital Medical University, Beijing, 100029, China. Broedl UC: Chemerin is associated with markers of inflammation and components of the metabolic syndrome but does not predict coronary Authors’ contributions XG carried out the molecular genetic studies and serum detection, atherosclerosis. Eur J Endocrinol 2009, 161:339-344. participated in the design of the study and drafted the manuscript. FZ 16. Gensini GG: A more meaningful scoring system for determining the carried out the immunoassays. XG, FZ and XZ involved in the acquisition of severity of coronary heart disease. Am J Cardiol 1983, 51:606. data as well as analysis and interpretation of data. YL and FG collected 17. Spiroglou SG, Kostopoulos CG, Varakis JN, Papadaki HH: Adipokines in adipose biopsy samples and serum samples. XG performed the statistical periaortic and epicardial adipose tissue: differential expression and analysis. HT and LW have been involved in revising the manuscript critically relation to atherosclerosis. J Atheroscler Thromb 2010, 17:115-130. for important intellectual content. HT, SM and FG conceived of the study, 18. Becker M, Rabe K, Lebherz C, Zugwurst J, Göke B, Parhofer KG, Lehrke M, and participated in its design and coordination. 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Baker AR, Silva NF, Quinn DW, Harte AL, Pagano D, Bonser RS, Kumar S, doi:10.1186/1475-2840-10-87 McTernan PG: Human epicardial adipose tissue expresses a pathogenic Cite this article as: Gao et al.: Association of chemerin mRNA expression profile of adipocytokines in patients with cardiovascular disease. in human epicardial adipose tissue with coronary atherosclerosis. Cardiovasc Diabetol 2006, 5:1. Cardiovascular Diabetology 2011 10:87. 13. Baker AR, Harte AL, Howell N, Pritlove DC, Ranasinghe AM, da Silva NF, Youssef EM, Khunti K, Davies MJ, Bonser RS, Kumar S, Pagano D, McTernan PG: Epicardial adipose tissue as a source of NFκB and JNK

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