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Authors
G. Iacobellis1, C. R. di Gioia2, M. Di Vito2, L. Petramala3, D. Cotesta3, V. De Santis4, D. Vitale4, L. Tritapepe4, C. Letizia3
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ epicardial adipose tissue ● ▶ epicardial fat ● ▶ adrenomedullin ● ▶ coronary artery disease ●
Abstract &
received 18.03.2009 accepted 15.06.2009 Bibliography DOI 10.1055/s-0029-1231081 Published online: July 21, 2009 Horm Metab Res 2009; 41: 855–860 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Prof. G. Iacobellis, MD, PhD Department of Medicine St. Joseph’s Hospital 50 Charlton Avenue East Fontbonne Bldg Hamilton Ontario L8N 4A6 Canada Tel.: + 1/905/521 1155 32739 Fax: + 1/905/521 6068
[email protected] [email protected]
The aim of the study was to test 1) whether chronic and stable coronary artery disease (CAD) could downregulate epicardial fat adrenomedullin synthesis and secretion, and decrease intracoronary plasma adrenomedullin levels, and 2) whether intracoronary plasma adrenomedullin levels could be related to epicardial adipose tissue adrenomedullin gene and protein expression in subjects with CAD. We examined 12 patients with CAD who required coronary artery bypass graft (CABG) and 10 patients with non-CAD who underwent cardiac surgery for valve replacement. Plasma levels of adrenomedullin were measured in peripheral vein circulation, in left coronary artery (LCA) and coronary sinus (CS) during coronary angiography. Epicardial adipose tissue biopsy for Reverse Transcription and RealTime PCR (RT-PCR) adrenomedullin mRNA analysis and Western Blotting (WB) protein expression
Introduction & It has been recently suggested that epicardial fat may play an active role in coronary artery disease (CAD) and atherosclerosis [1–14]. Epicardial fat is the true visceral fat depot of the heart and source of several bioactive adipokines [1–5]. Because of the close anatomical relationship to the heart and the absence of fascial boundaries epicardial adipose tissue may locally modulate the coronary arteries and myocardium through paracrine secretion of adipokines. It is plausible that local release of cytokines from periadventitial epicardial fat could traverse the coronary wall by diffusion from outside-to-inside or go directly into vasa vasorum and downstream into the arterial wall. However, a dual role of the epicardial adipose tissue has been suggested [2]. In fact human epicar-
was performed during cardiac surgery in all subjects. Peripheral, LCA, and CS plasma adrenomedullin levels were significantly lower in CAD patients than in those with non-CAD (3.0 ± 0.9 vs. 4.4 ± 0.9 pg/ml p < 0.01; 2.9 ± 1 vs. 4.05 ± 0.8 pg/ml, p < 0.01, 3.1 ± 0.9 vs. 3.98 ± 0.9 pg/ml p = 0.04, respectively). However, CS adrenomedullin levels were not statistically different than those in LCA suggesting that adrenomedullin was not secreted from epicardial fat into the coronary artery lumen. Epicardial fat adrenomedullin mRNA levels and protein expression were lower in patients with CAD than in those with non-CAD (p < 0.01 for both). We conclude that 1) epicardial fat adrenomedullin gene and protein expression can be downregulated in CAD subjects, and 2) intracoronary adrenomedullin levels are lower in CAD. No evidence that epicardial adipose tissue really contributes intracoronary adrenomedullin can be provided at this time.
dial fat has been shown to produce and secrete pro-, but also anti-inflammatory cytokines, like adiponectin and adrenomedullin. We have previously demonstrated that adiponectin is expressed by the epicardial adipose tissue and significantly correlates with intracoronary adiponectin levels [12, 13]. The latter finding was particularly supportive of the hypothesis of an interaction between the epicardial fat and the coronary circulation. Adrenomedullin has been indicated to have cardioprotective effects, as well as adiponectin. Adrenomedullin is a peptide with vasodilating, antioxidative, angiogenic, and anti-inflammatory properties [15]. Epicardial preadipocytes, which are part of the stromal cells and are more numerous in epicardial fat tissue, express adrenomedullin and its receptors, as recently described [14]. It has been suggested that adrenomedullin synthe-
Iacobellis G et al. Epicardial Fat and Intracoronary Adrenomedullin … Horm Metab Res 2009; 41: 855–860
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Epicardial Adipose Tissue and Intracoronary Adrenomedullin Levels in Coronary Artery Disease
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Study Design & This was a cross-sectional study conducted over one-year period. We examined a total of 22 individuals: 12 consecutive Caucasian patients with established CAD who required coronary artery bypass graft (CABG) intervention and a control group of 10 consecutive patients without CAD (non-CAD) who underwent cardiac surgery for aortic or mitral valve replacement. Epicardial adipose tissue biopsy for adrenomedullin protein extraction and gene expression was performed during cardiac surgery in overall CAD and non-CAD subjects. Plasma levels of adrenomedullin were measured in the peripheral vein circulation and in blood samples that were simultaneously collected from the left coronary artery (LCA) and coronary sinus (CS) during coronary angiography before cardiac surgery, in all subjects. Anthropometrics and fasting blood samples were also obtained before cardiac surgery in overall subjects.
Subjects and Methods & Subjects Study group (CAD) was formed by 12 patients (3 women, 9 men, mean age of 66 ± 10 years) with clinically and angiographically established CAD who underwent CABG. Control group (nonCAD) was formed by 10 subjects (7 women, 3 men, mean age of 65 ± 9 years) who underwent surgery for aortic or mitral valve replacement. Controls were randomly selected among patients who did not undergo CABG intervention. These patients had no history or clinical signs of CAD and showed normal coronary arteries on coronary angiography and normal ejection fraction on echocardiography. Patients with heart and renal failure, liver, acute or chronic infective or neoplastic diseases and who were taking corticosteroids, estrogens or psychotropic drugs were excluded from the study. This study was conducted in accordance with the guidelines proposed in The Declaration of Helsinki and has been approved by the ethical committee of ‘‘Sapienza University’’. All subjects gave their informed consent before surgery.
Methods & Anthropometrics Body Mass index (BMI) was calculated as body weight divided by height squared. Minimum waist circumference (minimum circumference between the lower rib margin and the iliac crest, midwaist) was measured in centimeters while the subjects were standing with their heels together.
Blood measurements & Plasma adrenomedullin was measured by a specific radioimmunoassay (RIA, Phoenix, Pharm. Inc, Mountain View, CA, USA), as previously described [20]. The anti-AM antibody cross-reacts 100 % with human AM-(1–52) but not with rat AM-(1–50), human amylin, human calcitonin gene-related peptide, endothelin-1, a-atrial natriuretic peptide (1–28), and b-atrial natriuretic peptide. Interassay variation was 12 % and intra-assay variation was 5 %. Fasting blood samples were obtained from overall subjects. Plasma glucose was determined by the glucose oxidase method [Autoanalyzer, Beckman Coulter, Inc., Fullerton, CA; coefficient of variation (CV), 1.9 ± 0.2 %]. Plasma total cholesterol (CV, 3.4 ± 0.2 %), high density lipoprotein cholesterol (HDL) (CV, 3.7 ± 0.4 %) and triglycerides (CV, 3.1 ± 0.5 %) concentrations were measured using enzymatic kits (Ortho-Clinical Diagnostic, Milan, Italy).
Coronary Angiography & Coronary angiography was performed according to the Judkins’ technique, and images of the coronary tree were obtained in routine, standardized projections with the digital quantitative Philips Integris 30 00 system in all patients. Two experienced cardiologists, who had no knowledge of the patients’ clinical characteristics and biochemical results, visually reviewed all angiographic images. Sampling from the coronary sinus (CS) was achieved through retrograde cannula (Medtronic® 15F retrograde coronary sinus perfusion cannula) inserted via right atrium before heparin administration. Coronary sinus cannulation was then used to deliver retrograde blood cardioplegia. The methods that we used to confirm CS cannulation involved distal pressure assessment at the catheter tip, oxygen saturation and transesophageal echocardiography view. A 5 ml of blood sample was drawn from CS after a discharge of the same quantity.
Epicardial adipose tissue collection Epicardial adipose tissue biopsy samples (average 0.5–1.0 g) were taken, before heparin administration, near the proximal tract of the right coronary artery. Each tissue sample was snap frozen in liquid nitrogen and stored at − 80 ° C until analysis.
Adrenomedullin Gene and Protein Expression & Reverse Transcription and Real-Time PCR analysis Reverse Transcription was performed using the High-Capacity cDNA Archive Kit (Applied Biosystem–Foster City, CA, USA) according to the manufacturer’s instruction. This was followed by quantitative real-time analysis, which was performed with
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sis from the epicardial fat could play a protective role on the coronary arteries [14]. However, the relationship of adrenomedullin with CAD is still partially contradictory and not completely understood. Some authors reported higher plasma adrenomedullin levels in CAD subjects [16–18] whereas others described lower adrenomedullin concentrations in these subjects [19]. Additionally, whether adrenomedullin expression from the epicardial fat may correlate with adrenomedullin levels in the coronary circulation is still unanswered. In this study we sought to test 1) whether chronic and stable (CAD) could downregulate epicardial fat adrenomedullin synthesis and secretion and be associated with decreased intracoronary plasma adrenomedullin levels, and 2) whether intracoronary plasma adrenomedullin levels could be related to epicardial adipose tissue adrenomedullin gene and protein expression in subjects with CAD.
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Western Blot Analysis Twenty μg of each samples were heat-denaturated for 10 min, loaded on NuPAGE 4–12 % Bis-Tris gel and run on ice at 200 V for 50 min using MOPS Buffer a 4 ° C (Invitrogen, Carlsbad, UK). Proteins were transferred, using iBlot Dry Blotting Sistem (Invitrogen, Carlsbad, UK), to a PVDF membrane (Invitrogen, Carlsbad, UK) at 20 V for 8 min. The membrane was blocked for 1 h in TBST and 5 % albumin, and incubated for 1 h at 4 ° C with a polyclonal antibody for ADM (dilution 1:1 000) that recognized the epitope corresponding to amino acids 1–85 representing full length ADM of human origin (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA). The membrane was washed with TBS-T and incubated for 1 h at room temperature with anti-rabbit IgG peroxidase-conjugated secondary antibody (dilution 1:10 000, SIGMA Saint Louis, Missouri, USA). Immunoreaction signals were detected by autoradiography (Kodak Biomax Light Film Sigma-Aldrich, St. Louis, MO, USA) using Chemiluminescent Peroxidase Substrate kit (Sigma-Aldrich, St. Louis, MO, USA). Monoclonal anti-beta actin antibody (dilution 1:10 000, Sigma-Aldrich, St. Louis, MO, USA) was used as control for equal loading and to normalized the immunoreactivity of ADM in each sample. Densitometric analysis of bands was performed using an Imaging Densitometric System (Quantity-One, Biorad, Hercules, CA, USA). Data are expressed in arbitrary units as ADM/actin ratio.
Statistical Analysis Data in the text and in the tables are expressed as mean ± Standard Deviation (SD). The sample size of this study provided us the statistical power of 80 % (1α = 0.05) to detect differences between CAD and non-CAD subjects. Comparisons between CAD and non-CAD subjects’ parameters were calculated by unpaired ttest. Independent correlates of intracoronary adrenomedullin were assessed by multiple linear regression analysis. A value of p < 0.05 was considered statistically significant.
Table 1 Clinical parameters in CAD and non-CAD subjects
Age (years) BMI (kg/m2) Waist circumference (cm) SBP (mmHg) DBP (mmHg) Glucose (mg/dl) Total cholesterol (mg/dl) HDL (mg/dl) Triglycerides (mg/dl) Type 2 Diabetes Dyslipidemia Hypertension Smoking Family history of CAD Unstable angina pectoris Stable angina pectoris 1-vessel CAD 2-vessel CAD 3-vessel CAD
CAD
Non-CAD
n = 12
n = 10
66 ± 10 22 ± 2.9 102 ± 9 130 ± 10 70 ± 5 108 ± 18 190 ± 25 46 ± 5 146 ± 45 3 9 5 4 1 2 10 0 1 11
65 ± 9 20 ± 1.6 85 ± 9 110 ± 10 70 ± 5 85 ± 16 197 ± 27 47 ± 4 116 ± 48 0 5 3 4 1 0 0 0 0 0
p-Value n. s. n. s. < 0.01 < 0.05 n. s. < 0.01 n. s. n. s. < 0.05
Data are expressed as mean ± SD. CAD: coronary artery diseases; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; Glucose: fasting glucose; HDL: high density lipoprotein cholesterol.
tor blockers or beta blockers). Three CAD subjects were taking metformin for type 2 diabetes with an optimal glucose control (HbA1c < 7 %). None within the two groups were taking diuretics. No significant differences in the smoking habit between the two groups occurred
Plasma Adrenomedullin levels in the Peripheral Vein Circulation Peripheral plasma adrenomedullin levels were significantly lower in CAD patients than in those with non-CAD (3.0 ± 0.9 vs. 4.4 ± 0.9 pg/ml, p < 0.01).
Plasma Adrenomedullin levels in the Coronary Circulation LCA and CS plasma adrenomedullin levels were significantly lower in CAD patients than in those with non-CAD (2.9 ± 1 vs. 4.05 ± 0.8 pg/ml, 3.1 ± 0.9 vs. 3.98 ± 0.9 pg/ml, p < 0.01 and p = 0.04, ▶ Fig. 1). Adrenomedullin levels in CS were slightly respectively (● higher than those in LCA (3.1 vs. 2.9 pg/ml) in CAD subjects, but the difference was not quite statistically significant.
Adrenomedullin mRNA levels in Epicardial Adipose Tissue RT-PCR analysis showed that adrenomedullin mRNA levels in epicardial adipose tissue were significantly lower in patients ▶ Fig. 2). with CAD than in those with non-CAD (p < 0.001) (●
Results & Clinical characteristics of the CAD and non-CAD subjects are summarized in Table 1. CAD subjects had higher waist circumference, fasting glucose, systolic blood pressure, and triglycerides than non-CAD subjects. No statistically significant differences in age, BMI, diastolic blood pressure, total and HDL cholesterol between the two groups were found. CAD and non-CAD did not significantly differ in lipid-lowering agents (statins) and antihypertensive medications (angiotensinogen-converting enzyme inhibitors, angiotensin II recep-
Adrenomedullin Protein Expression in Epicardial Adipose Tissue Adrenomedullin protein expression in epicardial adipose tissue was lower in patients with CAD than in those with non-CAD ▶ Fig. 3). (0.82 ± 0.35 vs. 1.48 ± 0.70 p < 0.01) (●
Iacobellis G et al. Epicardial Fat and Intracoronary Adrenomedullin … Horm Metab Res 2009; 41: 855–860
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the ABI Prism 7 900HT using 10 μl of the amplified cDNA aliquot. Predesigned Taqman primers and probes specific for adrenomedullin and housekeeping gene 18 S were used and PCR was performed using Universal PCR Master Mix®, no AmpErase® Ung. Thermal cycling conditions comprised an initial denaturation step at 95 ° C for 10 min and 40 cycles at 95 ° C for 15 s and 60 ° C for 1 min. All reagents were supplied by Applied Biosystems. Each sample was loaded in triplicate and a negative and a positive control was included in all plates. The expression levels of the analyzed genes were normalized to the level of the housekeeping gene. The housekeeping gene 18 S was provided as a preoptimized control probe by Applied Byosystem-Foster City, CA, USA. The housekeeping gene 18S is commonly used as control probe.
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A LCA
4.00
CS
2 1.8
Adrenomedullin/β Actin
plasma Adrenomedullin (pg/ml)
5.00 4.50 3.50 3.00 2.50 2.00 1.50 1.00 0.50
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2
0.00 CAD
0
non-CAD
Fig. 1 Intracoronary adrenomedullin levels. Left coronary artery [LCA, black column] and coronary sinus [CS, grey column] plasma adrenomedullin levels were significantly lower in CAD patients than in those with non-CAD (2.9 ± 1 vs. 4.05 ± 0.8 pg/ml, p < 0.01, 3.1 ± 0.9 vs. 3.98 ± 0.9 pg/ml, p = 0.04, respectively).
CAD
B
CAD
non-CAD
non-CAD
Adrenomedullin
Adrenomedullin mRNA 0.9 0.8 0.7 0.6
Fig. 3 Epicardial adipose tissue Adrenomedullin protein expression. Western Blotting (WB) analysis of adrenomedullin protein expression in epicardial adipose tissue, expressed as adrenomedullin/actin ratio (panel A); representative WB data in a sample of two CAD and non-CAD subjects (panel B). Adrenomedullin protein expression was lower in patients with CAD than in those with non-CAD (0.82 ± 0.35 vs. 1.48 ± 0.70 p < 0.01).
0.5 0.4 0.3 0.2 0.1 CAD
non-CAD
Fig. 2 Epicardial adipose tissue adrenomedullin mRNA levels. RT-PCR adrenomedullin analysis mRNA in epicardial adipose tissue: dot-scatter plot graphic showing adrenomedullin mRNA levels were significantly lower in patients with CAD than in those with non-CAD (p < 0.001).
Correlates of Intracoronary Plasma Adrenomedullin levels In a multivariate model, peripheral adrenomedullin levels and adrenomedullin mRNA levels were the best correlates of LCA adrenomedullin levels, R2 = 0.75, p = 0.01, p = 0.04 respectively. When CS adrenomedullin was considered as dependent variable, peripheral adrenomedullin was its only independent and significant correlate. These correlations were substantially unchanged when adjusted by gender and when CAD and nonCAD subjects were pooled together.
Discussion & In addition to its detrimental effect on the heart and coronary arteries, epicardial fat may display cardioprotective properties through local secretion of protective adipokines, such as adiponectin and adrenomedullin. However, not much attention has been focused on the malfunction of the suggested cardioprotective role of the epicardial fat. In this study, our first hypothesis was that chronic CAD could affect local and systemic adrenomedullin plasma levels and also
adrenomedullin synthesis and production from the epicardial fat. Our results seem to support this first hypothesis. In fact, in this study intracoronary adrenomedullin concentrations were significantly lower in CAD subjects than those in nonCAD individuals. The relationship of adrenomedullin with CAD is still partially unclear. It has been reported that peripheral plasma adrenomedullin levels are upregulated during and following acute myocardial infarction [16, 17] and play a role in modulating myocardial tolerance to ischemia-reperfusion injury [18]. However, our findings are consistent with a previous observation showing lower adrenomedullin levels in the coronary sinus of CAD subjects when compared to control subjects [19]. Decreased adrenomedullin levels in the coronary circulation may be related to the formation and progression of coronary artery lesions in patients with CAD. Increase in intracoronary adrenomedullin levels have been reported only with improved hemodynamic conditions, such as after coronary revascularization [21], as we previously observed for intracoronary adiponectin [22]. Intracoronary coronary adrenomedullin could be also low probably because the adrenomedullin in the venous blood returned to the heart and then pumped out could be low in CAD subjects. In this study, we found that also adrenomedullin protein and gene expression in epicardial fat are significantly lower in CAD patients than those in subjects without CAD. It has been suggested that epicardial fat adrenomedullin synthesis increases in response to chronic hypoxemia [14]. However, we may differently hypothesize that this adaptive response to local insult may be impaired in long-term and stable CAD. For unknown reasons, epicardial fat cardioprotective role may be malfunctioning under pathological circumstances. Likely, the balance of different mediators under pathophysiological conditions, as well as coronary status, co-morbidities, age or body fatness, is of importance. Secretion of epicardial fat adrenomedullin in the coronary circu-
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β-Actin
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This study has some limitations and caution might be necessary in interpreting the results. No conclusions on pathophysiological mechanisms can be drawn from this study. The sample size of the present study was relatively small and further investigations in larger populations are required to confirm our data. However, studies of this specific nature frequently have similar sample size and also ethical concerns prevented us from obtaining epicardial adipose tissue biopsy samples in patients after cardiac surgery. Larger noncardiac visceral adipose tissue depots have not been considered in this study. Although, the production of adrenomedullin by larger fat depots can be considered, we believe that the epicardial fat is unique in playing a direct role on the coronary circulation. Concomitant pharmacological treatments could interfere with study results. Gender distribution was different between the two groups, but correlations were unchanged when adjusted by the sex.
Acknowledgements & We are thankful to Giovanni Clemente for his excellent technical contribution. Affiliations 1 Department of Medicine, McMaster University, Hamilton, ON, Canada 2 Department of Experimental Medicine, Sapienza University, Rome, Italy 3 Department of Clinical Sciences, Sapienza University, Rome, Italy 4 Department of Anesthesiology and Intensive Care, Sapienza University, Rome, Italy
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lation could be downregulated and therefore lower than proinflammatory adipokine production in subjects with established CAD. On the other hand, adrenomedullin mRNA levels could be upregulated only in response to certain stimuli that may occur with improved hemodynamic conditions. In addition, it is interesting to underline that adrenomedullin amount from the coronary plaque was found significantly higher in patients with unstable angina pectoris than in those with stable angina pectoris [23]. Given that the majority of our CAD subjects had stable angina, this may also explain the lower epicardial fat adrenomedullin synthesis and expression. Adrenomedullin mRNA and protein over-expression may occur only in patients with unstable CAD or acute cardiac events, such as after myocardial infarction. However, the factors responsible for the down or upregulation of adrenomedullin synthesis and secretion are probably more complex and not completely understood. Our results seem to be in disagreement with the increased adrenomedullin mRNA levels in epicardial adipose tissue in CAD patients, reported by Silaghi et al. [14]. We may consider some differences in the study population between the two studies. While metabolic control, gender distribution and adiposity phenotypes did not substantially differ, our subjects are older and significantly leaner than those evaluated by Silaghi et al. [14]. It is plausible that variation in age and body fatness may influence adipose tissue pathophysiology. Our second hypothesis was that intracoronary plasma adrenomedullin levels could be related and modulated by the epicardial adipose tissue. Whether epicardial adipose tissue could be a source of intracoronary adrenomedullin has been suggested, but not clearly demonstrated. Our hypothesis was based on the fact that because epicardial adipose tissue lacks fascia and shares the same microcirculation with the myocardium, adrenomedullin expression from epicardial fat might participate in intracoronary adrenomedullin levels. Paracrine and/or vasocrine pathways for this local interaction have been suggested [5]. To test this hypothesis adrenomedullin levels were collected and measured in the coronary sinus into which adrenomedullin is physiologically secreted, although myocardial adrenomedullin production should be reliably calculated with measurement of blood flow in all the coronary arteries, as Yasu et al. stated in their study [16]. If adrenomedullin was secreted transmurally from epicardial fat into the coronary artery lumen, adrenomedullin levels in coronary sinus should have been higher than those in arterial blood. Nevertheless, our study does not support this hypothesis. Our data show that CS adrenomedullin levels are marginally, but not significantly higher than those in the coronary artery. However, even if the adrenomedullin levels in the CS were significantly higher, this would be not sufficient to affirm that the epicardial fat really contributes to the intracoronary adrenomedullin. In fact, we know that the CS receives blood from the veins which drain both the epicardial fat and the myocardium. The myocardium may be the source of the adrenomedullin, either from the myocytes or the fibroblasts in the heart. Obviously, because there is no significant increase in CS adrenomedullin levels, the epicardial fat cannot be considered the source of intracoronary adrenomedullin. In summary, this study suggests that chronic and stable CAD is associated with low intracoronaric and systemic adrenomedullin levels and also downregulates epicardial fat adrenomedullin gene and protein expression. No evidence that epicardial adipose tissue really contributes to intracoronary adrenomedullin concentration can be provided at this time.
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Iacobellis G et al. Epicardial Fat and Intracoronary Adrenomedullin … Horm Metab Res 2009; 41: 855–860
