Nutrition, Metabolism & Cardiovascular Diseases (2014) xx, 1e7
Available online at www.sciencedirect.com
Nutrition, Metabolism & Cardiovascular Diseases journal homepage: www.elsevier.com/locate/nmcd
Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease E. Dozio a,*, S. Briganti b, E. Vianello a, G. Dogliotti a,1, A. Barassi c, A.E. Malavazos b, F. Ermetici b, L. Morricone b, A. Sigruener d, G. Schmitz d, M.M. Corsi Romanelli a,e a
Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy Diabetology and Metabolic Diseases Unit, I.R.C.C.S. Policlinico San Donato, San Donato Milanese, Milan, Italy c Department of Sciences for Health, Ospedale San Paolo, Università degli Studi di Milano, Milan, Italy d Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany e Service of Laboratory Medicine 1 e Clinical Pathology, Department of Health Services of Diagnosis and Treatment e Laboratory Medicine, I.R.C.C.S. Policlinico San Donato, San Donato Milanese, Milan, Italy b
Received 30 May 2014; received in revised form 6 August 2014; accepted 29 August 2014 Available online - - -
KEYWORDS Coronary artery disease; Epicardial adipose tissue; Inflammation; Vitamin D; Vitamin D receptor; CYP27B1; CYP24A1
Abstract Background and aims: Alterations in epicardial adipose tissue (EAT) biology (i.e. increased fat thickness and inflammation) have been described in coronary artery disease (CAD) patients. In addition to its classic role in the regulation of calcium-phosphate homeostasis, vitamin D may exert immune-regulatory and anti-inflammatory effects. Whether EAT inflammation may be linked to vitamin D deficiency is still unknown. In the present study we evaluated plasma 25-hydroxycholecalciferol (25OHD) level in CAD patients and its relationship with EAT ability to locally metabolize vitamin D, EAT expression of inflammation-related molecules and EAT thickness. Methods and results: Plasma 25OHD level was quantified by an immunoluminometric assay. EAT expression of inflammation-related molecules (MCP-1, PTX3, TNFa, IL-6, adiponectin), vitamin D r e c e p t o r ( V D R ) , C Y P 2 7 B 1 ( 2 5 O H D - a c t i v a t i n g e n z y m e ) a n d C Y P 2 4 A 1 ( 1, 2 5 dihydroxycholecalciferol-metabolizing enzyme) was performed by microarray. EAT thickness was quantified by echocardiography. Median plasma 25OHD level was 10.85 ng/mL and 83% of CAD patients displayed 25OHD level below 20 ng/mL. At decreasing plasma 25OHD concentration, we observed a down-regulation in CYP27B1 and CYP24A1 level and an increased expression of VDR and pro-inflammatory cytokines (MCP-1, PTX3, TNFa, IL-6) at EAT level. No correlation was observed between plasma 25OHD level and EAT thickness. Conclusion: Our data suggest an increased activation of inflammatory pathways at EAT level possibly related to systemic and local vitamin D deficiency in CAD patients. Whether maintaining an optimal vitamin D status may be helpful to reduce EAT inflammation and to prevent CAD and its progression needs further investigation. ª 2014 Elsevier B.V. All rights reserved.
* Corresponding author. Department of Biomedical Sciences for Health, Università degli Studi di Milano, Via Luigi Mangiagalli 31, 20133, Milan, Italy. Tel.: þ39 02 50315342; fax: þ39 02 50315338. E-mail address: [email protected] (E. Dozio). 1 She actually has a new appointment at the Institute of Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany. http://dx.doi.org/10.1016/j.numecd.2014.08.012 0939-4753/ª 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
2
Introduction Epicardial fat (EAT) is a metabolically active visceral adipose tissue which may locally interact with myocardium and coronary arteries through vasocrine and paracrine secretion of various adipokines and cytokines [1e4]. Recent reports indicated that EAT accumulation may be a risk factor for coronary artery disease (CAD) and alterations in EAT biology, such as increased thickness, elevated inflammatory infiltrate and cytokine production, have been observed in CAD patients [1,3]. Vitamin D is a fat-soluble hormone able to regulate the transcription of many genes through vitamin D receptor (VDR) [5]. The major circulating form of vitamin D is 25hydroxycholecalciferol (25OHD) which, prior to binding to VDR, needs to be hydroxylated by the 1a-hydroxylase CYP27B1 into the biologically active form 1,25dihydroxycholecalciferol (calcitriol) which is further degraded by the 24-hydroxylase CYP24A1 [6]. Recent studies suggested that vitamin D has multiple functions in addition to its well known role in the regulation of calcium-phosphate homeostasis and adipose tissue has been recognized as a target for vitamin D actions [7]. In particular, adipocytes may be directly involved in the local synthesis and degradation of calcitriol which is able to affect adipocyte biology by modulating pre-adipocyte differentiation, lipid accumulation and mobilization and also adipokine production (reviewed in Ref. [8]). Vitamin D may also exert immune-regolatory effects [9] and suppress the production of different pro-inflammatory cytokines in several cell types [10e12]. In the field of human adipose tissue there are only few studies which investigated the potential anti-inflammatory role of vitamin D. Most of these utilized in vitro cultures of adipocytes and created an in vitro artificial inflammatory state [13e15]. Only one study conducted in hypercholesterolemic swine explored the association between vitamin D deficiency and inflammation at EAT level [16]. Today it is unknown whether the increased inflammation observed at EAT level in CAD patients may be linked to vitamin D deficiency. In the present study we aimed to evaluate plasma 25OHD level in CAD patients and its relationship with EAT ability to locally metabolize vitamin D, EAT expression of inflammation-related molecules and EAT thickness.
E. Dozio et al.
3% variation in body weight in the previous 3 months. Patients were measured for height, weight, waist and hip circumferences; body mass index (BMI) and waist/hip ratio (WHR) were calculated. Medication intake was also recorded. The study protocol was approved by the local Ethics Committee (ASL Milano Due, n 2516) and patients gave their written informed consent to the examination protocol, conducted in accordance with the Declaration of Helsinki, as revised in 2000. Blood collection Blood samples were collected after an overnight fasting into pyrogen-free tubes with ethylendiaminetetraacetic acid as anticoagulant. Plasma samples were separated after centrifugation at 1000 g for 15 min and were stored at 20 C until analysis. Fasting glucose, insulin, total- and HDL-cholesterol, triglycerides and CRP were assayed as previously reported [4,17]. LDL-cholesterol was calculated with the Friedewald formula. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following equation: HOMA-IR Z fasting insulin [mU/mL] fasting glucose [mmol/L]/22.5. EAT quantification All patients were examined by echocardiography using an M-mode color-Doppler VSF (Vingmed-System Five; General Electric, Horten, Norway) with a 2.5- to 3.5-MHz transducer probe. EAT thickness was measured on the free wall of the right ventricle from both parasternal long- and short-axis views and appears as a non-homogeneous echo-free space, as previously reported [18]. We chose to measure EAT thickness on the right ventricle for two reasons: (1) this point is recognized as the greatest absolute EAT thickness and (2) parasternal long- and short-axis views allow the most accurate measurement of EAT on the right ventricle with optimal cursor beam orientation in each view. EAT collection
Methods
EAT biopsy samples were harvested adjacent to the proximal right coronary artery prior to initiation of cardiopulmonary bypass pumping. Samples were stored in Allprotect Tissue Reagent (Qiagen, Hilden, Germany) at 20 C until RNA extraction.
Study population
RNA extraction and gene expression analysis
A total of 54 male CAD patients undergoing coronary artery bypass grafting surgery at the I.R.C.C.S. Policlinico San Donato were enrolled from October 2011 to March 2012. Patients were selected from a larger group according to the following exclusion criteria: 1) the known presence of generalized bone diseases, including hyperparathyroidism, rheumatoid arthritis, Cushing syndrome; 2) recent acute myocardial infarction, malignant disease, recent major abdominal surgery, renal and liver diseases; 3) more than
Total RNA was extracted from tissue with the RNeasy Lipid Tissue Kit according to the manufacturer’s procedure (Qiagen). RNA concentration was quantified by NanoDrop 2000 (ThermoScientific, Wilmington, Germany) and RNA integrity was assessed using the Agilent RNA 6000 Nano kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Gene expression analysis was performed by one color microarray platform (Agilent). 50 ng of total RNA was labeled with Cy3 using the Agilent LowInput
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
Epicardial adipose tissue inflammation is related to vitamin D deficiency
Quick-Amp Labeling kit-1 color, according to manufacturer’s instructions. cRNA was purified with the RNeasy Mini Kit (Qiagen) and the amount and labeling efficiency were measured with NanoDrop. Hybridization was performed using Agilent Gene Expression Hybridization Kit and scanning with Agilent G2565CA Microarray Scanner System. Data were processed using Agilent Feature Extraction Software (10.7) with the single color gene expression protocol and raw data were analyzed with ChipInspector Software (Genomatix, Munich, Germany). In brief, raw data were normalized on single probe level based on the array mean intensities and statistics were calculated based on the SAM algorithm by Tusher [19]. Fold changes were determined from normalized data. Immunoassays The quantitative determination of plasma 25OHD concentration was performed by a chemiluminescent immunoassay technology, according to manufacturer’s instructions (LIAISON, DiaSorin, Vercelli, Italy). Since only in two CAD patients 25OHD concentration was > of the cut off value of 30 ng/mL, sometimes used for optimal vitamin status, patients were classified according to 25OHD values reported by Thacher et al. [20]: 25OHD deficiency, 20 ng/mL. Statistical analysis Data are expressed as median and 25the75th percentiles or mean SD or number and percentage. The normality of data distribution was assessed by the KolmogoroveSmirnoff test. Fisher’s exact test was used for categorical outcomes. Differences across the three groups of 25OHD level were compared by one-way ANOVA followed by Tukey post-hoc test (for normally distributed parameters) or KruskaleWallis test followed by Dunn post-hoc test (for non-normally distributed parameters). Associations between parameters were examined by the Spearman correlation test. Data were analyzed using GraphPad Prism 5.0 biochemical statistical package (GraphPad Software, San Diego, CA). A p value 30 ng/mL. After patient stratification according to 25OHD level, the anthropometric parameters BMI, waist circumference, WHR as well as EAT
Reduced plasma 25OHD level is associated with increased VDR and reduced CYP27B1 and CYP24A1 expression at EAT level CAD patients were stratified according to plasma 25OHD level. Compared to 25OHD sufficient group, 25OHD deficient group displayed about a 2-fold increase in VDR expression (p < 0.05) (Fig. 1). A statistically significant reduction in CYP27B1 expression was observed in both 25OHD insufficient and deficient groups (p < 0.05 for both), as compared with 25OHD sufficient group (Fig. 1). CYP24A1 expression was also
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
4
E. Dozio et al.
Table 2 Demographic, anthropometric and biochemical characteristics of coronary artery disease patients classified according to plasma 25OHD level.
Age (years) Weight (Kg) BMI (kg/m2) Waist (cm) Hip (cm) WHR EAT thickness (mm) Fasting glucose (mg/dl) Fasting insulin (mU/ml) HOMA-IR Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) CRP (mg/dl) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) 25OHD (ng/mL)
25OHD 20 ng/mL
(n Z 24)
(n Z 21)
(n Z 9)
66.00, 58.25e72.50 74.00, 70.00e81.00 27.17, 25.13e28.63 104.00, 100.00e108.00 101.00, 91.75e106.00 1.02, 1.02e1.06 8.00, 6.25e9.25 82.50, 78.50e101.80 7.71, 5.08e13.51 1.50, 1.06e2.82 140.00, 129.00e167.00 34.00, 30.00e43.50 81.80, 70.85e109.00 117.00, 85.50e187.00 0.30, 0.20e1.00 120.00, 120.00e130.00 70.00, 70.00e70.00 6.88, 4.56e8.24a,b
66.00, 61.00e76.00 80.00, 69.00e90.50 27.75, 23.76e29.55 103.50, 92.50e114.00 105.00, 96.00e108.00 1.02, 0.99e1.06 9.00, 6.00e10.25 88.00, 75.25e116.30 6.75, 4.99e11.20 1.61, 1.07e2.61 138.50, 114.30e161.50 33.00, 25.50e42 77.20, 60.80e88.70 97.00, 75.50e143.00 0.45, 0.10e1.50 120.00, 112.50e127.50 70.00, 70.00e70.00 14.35, 11.64e16.74b
68.00, 58.50e75.00 78.00, 70.50e80.50 25.60, 24.43e28.74 102.00, 96.50e106.00 102.00, 96.50e107.00 1.02, 0.91e1.07 7.00, 6.00e8.50 80.00, 77.00e101.50 8.53, 4.91e13.43 2.16, 1.06e2.88 150.00, 117.50e167.50 44.00, 38.50e45.00 73.00, 58.20e104.10 104.00, 100e153.00 0.10, 1.10e1.20 130.00, 120.00e140.00 70.00, 70.00e80.00 23.70, 20.53e28.81
BMI: body mass index; CRP: C-reactive protein; EAT: epicardial adipose tissue; HOMA-IR: homeostatis model of insulin resistance; 25OHD: 25hydroxycholecalciferol; WHR: waist to hip ratio. Data are expressed as median and 25the75th percentiles. a p < 0.001 vs. 25OHD 10e20 ng/mL; b p < 0.001 vs. 25OHD >20 ng/mL (ANOVA).
decreased both in 25OHD deficient and insufficient groups (p < 0.05 and p < 0.01, respectively) (Fig. 1). Correlation analyses indicated that plasma 25OHD level was inversely correlated with VDR (r Z 0.48, p < 0.05) and positively with CYP27B1 (r Z 0.61, p < 0.001) and CYP24A1 (r Z 0.53, p < 0.05) expression at EAT level. Reduced plasma 25OHD level is associated to increased expression of inflammation-related molecules at EAT level CAD patients were stratified according to plasma 25OHD level. Compared to 25OHD sufficient group, 25OHD insufficient and deficient groups displayed about a 3-fold
increase in MCP-1 level (p < 0.05) (Fig. 2). A 2-fold increase in PTX-3 level has also been observed in the 25OHD deficient group, as compared with 25OHD sufficient group (p < 0.05) (Fig. 2). Expression of TNFa and IL-6 was up-regulated of about 2times in both insufficient and deficient groups, although these differences did not reach the statistical significance. Adiponectin level was almost the same in the three groups. Increased level of inflammation-related molecules is associated to increased VDR expression at EAT level CAD patients were further stratified according to tertiles (Q1-Q2-Q3) of VDR expression at EAT level.
Figure 1 Evaluation of VDR, CYP27B1 and CYP24A1 gene expression at EAT level according to plasma 25OHD concentration. CAD patients were stratified into three groups (sufficient, insufficient and deficient) according to plasma 25OHD concentration. Gene expression of VDR, CYP27B1 and CYP24A1 at EAT level is expressed as fold change SD vs. 25OHD sufficient group. Normalized raw data (mean SD) are also reported. *p < 0.05, p < 0.01 vs. 25OHD sufficient group.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
Epicardial adipose tissue inflammation is related to vitamin D deficiency
5
Figure 2 Gene expression of inflammation-related molecules at EAT level according to plasma 25OHD concentration. CAD patients were stratified into three groups (sufficient, insufficient and deficient) according to plasma 25OHD concentration. Gene expression of MCP-1, PTX3, TNFa, IL-6 and adiponectin at EAT level is expressed as fold change SD vs. 25OHD sufficient group. Normalized raw data (mean SD) are also reported. *p < 0.05 vs. 25OHD sufficient group.
Compared to Q1, MCP-1 and TNFa expression in Q2 was about 2.5-fold higher (p < 0.05, for both) (Fig. 3). We also observed about a 3-fold increase in MCP-1 (p < 0.05), a 2fold increase in PTX-3 (p < 0.05), a 2.5-fold increase in TNFa (p < 0.05) and a six-fold increase in IL-6 levels (p < 0.05) in Q3 compared to Q1 (Fig. 3). Adiponectin level was almost the same in the three groups. Correlation analyses also indicated a positive correlation between VDR and MCP-1 (r Z 0.69, p < 0.001), PTX-3 (r Z 0.45, p < 0.05), TNFa (r Z 0.49, p < 0.05) and IL-6 (r Z 0.66, p < 0.01) expression at EAT level, but not for adiponectin (r Z 0.27, p Z 0.24).
Discussion The novelty of our study is the observation that in CAD patients the expression of pro-inflammatory molecules at EAT level seems to be related to plasma 25OHD concentration and local expression of VDR. In fact, as 25OHD level decreases, local EAT expression of both VDR and proinflammatory cytokines increases. Moreover, patients with increased VDR expression also displayed higher level of pro-inflammatory mediators.
In addition to its well known role as a regulator of calcium homeostasis and bone metabolism, vitamin D may exert other different physiological functions. In particular, due to its ability to modulate T-lymphocyte proliferation and function and to suppress the production of inflammatory cytokines, vitamin D is recognized as a potential anti-inflammatory molecule [21e24]. Previous in vitro studies showed that vitamin D is able to down regulate the expression of pro-inflammatory mediators in human monocytes stimulated with interferon-g [11] and to inhibit the production of MCP-1 and other pro-inflammatory mediators in human preadipocytes [13] and mature adipocytes [14]. Although these previous studies suggested that vitamin D may protect against adipose tissue inflammation by disrupting the deleterious cycle of macrophage recruitment, our study represent the first human study on the potential association between vitamin D and inflammation at EAT level in CAD patients. As previously observed for other adipose depots, i.e. subcutaneous and visceral [25], also EAT expresses the activating 1ahydroxylase enzyme CYP27B1, the degrading 24hydroxylase enzyme CYP24A1 and VDR, thus suggesting the existence of a paracrineeautocrine effect of vitamin D at EAT level.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
6
E. Dozio et al.
Figure 3 Gene expression of inflammation-related molecules at EAT level according to tertiles of VDR expression. CAD patients stratified according to tertiles (Q1-Q2-Q3) of VDR expression at EAT level. Gene expression of MCP-1, PTX3, TNFa, IL-6 and adiponectin at EAT level is expressed as fold change SD vs. Q1. Normalized raw data (mean SD) are also reported. *p < 0.05.
According to previous reports, our data seem to suggest that vitamin D may be involved in the regulation of inflammation also at EAT. In fact, in condition of plasma 25OHD insufficiency and deficiency, the local vitamin D metabolism seems to be suppressed and associated to an increased production of inflammatory mediators. We have also observed an increased expression of VDR associated both to reduced plasma 25OHD level as well as to increased EAT inflammation. Recent studies suggested that VDR expression may be up-regulated in adipocytes after exposure to macrophages-derived products [26] and in visceral fat VDR level was higher in obese than lean women [25]. Our data do not directly prove the molecular mechanisms inducing VDR up-regulation, anyway, given the potential anti-inflammatory action of vitamin D, this could be a counter-regulatory response to ameliorate inflammation. The open question is whether vitamin D supplementation could be a useful tool to counteract local EAT inflammation in these patients. The prevalence of hypovitaminosis D (
Available online at www.sciencedirect.com
Nutrition, Metabolism & Cardiovascular Diseases journal homepage: www.elsevier.com/locate/nmcd
Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease E. Dozio a,*, S. Briganti b, E. Vianello a, G. Dogliotti a,1, A. Barassi c, A.E. Malavazos b, F. Ermetici b, L. Morricone b, A. Sigruener d, G. Schmitz d, M.M. Corsi Romanelli a,e a
Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy Diabetology and Metabolic Diseases Unit, I.R.C.C.S. Policlinico San Donato, San Donato Milanese, Milan, Italy c Department of Sciences for Health, Ospedale San Paolo, Università degli Studi di Milano, Milan, Italy d Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany e Service of Laboratory Medicine 1 e Clinical Pathology, Department of Health Services of Diagnosis and Treatment e Laboratory Medicine, I.R.C.C.S. Policlinico San Donato, San Donato Milanese, Milan, Italy b
Received 30 May 2014; received in revised form 6 August 2014; accepted 29 August 2014 Available online - - -
KEYWORDS Coronary artery disease; Epicardial adipose tissue; Inflammation; Vitamin D; Vitamin D receptor; CYP27B1; CYP24A1
Abstract Background and aims: Alterations in epicardial adipose tissue (EAT) biology (i.e. increased fat thickness and inflammation) have been described in coronary artery disease (CAD) patients. In addition to its classic role in the regulation of calcium-phosphate homeostasis, vitamin D may exert immune-regulatory and anti-inflammatory effects. Whether EAT inflammation may be linked to vitamin D deficiency is still unknown. In the present study we evaluated plasma 25-hydroxycholecalciferol (25OHD) level in CAD patients and its relationship with EAT ability to locally metabolize vitamin D, EAT expression of inflammation-related molecules and EAT thickness. Methods and results: Plasma 25OHD level was quantified by an immunoluminometric assay. EAT expression of inflammation-related molecules (MCP-1, PTX3, TNFa, IL-6, adiponectin), vitamin D r e c e p t o r ( V D R ) , C Y P 2 7 B 1 ( 2 5 O H D - a c t i v a t i n g e n z y m e ) a n d C Y P 2 4 A 1 ( 1, 2 5 dihydroxycholecalciferol-metabolizing enzyme) was performed by microarray. EAT thickness was quantified by echocardiography. Median plasma 25OHD level was 10.85 ng/mL and 83% of CAD patients displayed 25OHD level below 20 ng/mL. At decreasing plasma 25OHD concentration, we observed a down-regulation in CYP27B1 and CYP24A1 level and an increased expression of VDR and pro-inflammatory cytokines (MCP-1, PTX3, TNFa, IL-6) at EAT level. No correlation was observed between plasma 25OHD level and EAT thickness. Conclusion: Our data suggest an increased activation of inflammatory pathways at EAT level possibly related to systemic and local vitamin D deficiency in CAD patients. Whether maintaining an optimal vitamin D status may be helpful to reduce EAT inflammation and to prevent CAD and its progression needs further investigation. ª 2014 Elsevier B.V. All rights reserved.
* Corresponding author. Department of Biomedical Sciences for Health, Università degli Studi di Milano, Via Luigi Mangiagalli 31, 20133, Milan, Italy. Tel.: þ39 02 50315342; fax: þ39 02 50315338. E-mail address: [email protected] (E. Dozio). 1 She actually has a new appointment at the Institute of Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany. http://dx.doi.org/10.1016/j.numecd.2014.08.012 0939-4753/ª 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
2
Introduction Epicardial fat (EAT) is a metabolically active visceral adipose tissue which may locally interact with myocardium and coronary arteries through vasocrine and paracrine secretion of various adipokines and cytokines [1e4]. Recent reports indicated that EAT accumulation may be a risk factor for coronary artery disease (CAD) and alterations in EAT biology, such as increased thickness, elevated inflammatory infiltrate and cytokine production, have been observed in CAD patients [1,3]. Vitamin D is a fat-soluble hormone able to regulate the transcription of many genes through vitamin D receptor (VDR) [5]. The major circulating form of vitamin D is 25hydroxycholecalciferol (25OHD) which, prior to binding to VDR, needs to be hydroxylated by the 1a-hydroxylase CYP27B1 into the biologically active form 1,25dihydroxycholecalciferol (calcitriol) which is further degraded by the 24-hydroxylase CYP24A1 [6]. Recent studies suggested that vitamin D has multiple functions in addition to its well known role in the regulation of calcium-phosphate homeostasis and adipose tissue has been recognized as a target for vitamin D actions [7]. In particular, adipocytes may be directly involved in the local synthesis and degradation of calcitriol which is able to affect adipocyte biology by modulating pre-adipocyte differentiation, lipid accumulation and mobilization and also adipokine production (reviewed in Ref. [8]). Vitamin D may also exert immune-regolatory effects [9] and suppress the production of different pro-inflammatory cytokines in several cell types [10e12]. In the field of human adipose tissue there are only few studies which investigated the potential anti-inflammatory role of vitamin D. Most of these utilized in vitro cultures of adipocytes and created an in vitro artificial inflammatory state [13e15]. Only one study conducted in hypercholesterolemic swine explored the association between vitamin D deficiency and inflammation at EAT level [16]. Today it is unknown whether the increased inflammation observed at EAT level in CAD patients may be linked to vitamin D deficiency. In the present study we aimed to evaluate plasma 25OHD level in CAD patients and its relationship with EAT ability to locally metabolize vitamin D, EAT expression of inflammation-related molecules and EAT thickness.
E. Dozio et al.
3% variation in body weight in the previous 3 months. Patients were measured for height, weight, waist and hip circumferences; body mass index (BMI) and waist/hip ratio (WHR) were calculated. Medication intake was also recorded. The study protocol was approved by the local Ethics Committee (ASL Milano Due, n 2516) and patients gave their written informed consent to the examination protocol, conducted in accordance with the Declaration of Helsinki, as revised in 2000. Blood collection Blood samples were collected after an overnight fasting into pyrogen-free tubes with ethylendiaminetetraacetic acid as anticoagulant. Plasma samples were separated after centrifugation at 1000 g for 15 min and were stored at 20 C until analysis. Fasting glucose, insulin, total- and HDL-cholesterol, triglycerides and CRP were assayed as previously reported [4,17]. LDL-cholesterol was calculated with the Friedewald formula. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following equation: HOMA-IR Z fasting insulin [mU/mL] fasting glucose [mmol/L]/22.5. EAT quantification All patients were examined by echocardiography using an M-mode color-Doppler VSF (Vingmed-System Five; General Electric, Horten, Norway) with a 2.5- to 3.5-MHz transducer probe. EAT thickness was measured on the free wall of the right ventricle from both parasternal long- and short-axis views and appears as a non-homogeneous echo-free space, as previously reported [18]. We chose to measure EAT thickness on the right ventricle for two reasons: (1) this point is recognized as the greatest absolute EAT thickness and (2) parasternal long- and short-axis views allow the most accurate measurement of EAT on the right ventricle with optimal cursor beam orientation in each view. EAT collection
Methods
EAT biopsy samples were harvested adjacent to the proximal right coronary artery prior to initiation of cardiopulmonary bypass pumping. Samples were stored in Allprotect Tissue Reagent (Qiagen, Hilden, Germany) at 20 C until RNA extraction.
Study population
RNA extraction and gene expression analysis
A total of 54 male CAD patients undergoing coronary artery bypass grafting surgery at the I.R.C.C.S. Policlinico San Donato were enrolled from October 2011 to March 2012. Patients were selected from a larger group according to the following exclusion criteria: 1) the known presence of generalized bone diseases, including hyperparathyroidism, rheumatoid arthritis, Cushing syndrome; 2) recent acute myocardial infarction, malignant disease, recent major abdominal surgery, renal and liver diseases; 3) more than
Total RNA was extracted from tissue with the RNeasy Lipid Tissue Kit according to the manufacturer’s procedure (Qiagen). RNA concentration was quantified by NanoDrop 2000 (ThermoScientific, Wilmington, Germany) and RNA integrity was assessed using the Agilent RNA 6000 Nano kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Gene expression analysis was performed by one color microarray platform (Agilent). 50 ng of total RNA was labeled with Cy3 using the Agilent LowInput
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
Epicardial adipose tissue inflammation is related to vitamin D deficiency
Quick-Amp Labeling kit-1 color, according to manufacturer’s instructions. cRNA was purified with the RNeasy Mini Kit (Qiagen) and the amount and labeling efficiency were measured with NanoDrop. Hybridization was performed using Agilent Gene Expression Hybridization Kit and scanning with Agilent G2565CA Microarray Scanner System. Data were processed using Agilent Feature Extraction Software (10.7) with the single color gene expression protocol and raw data were analyzed with ChipInspector Software (Genomatix, Munich, Germany). In brief, raw data were normalized on single probe level based on the array mean intensities and statistics were calculated based on the SAM algorithm by Tusher [19]. Fold changes were determined from normalized data. Immunoassays The quantitative determination of plasma 25OHD concentration was performed by a chemiluminescent immunoassay technology, according to manufacturer’s instructions (LIAISON, DiaSorin, Vercelli, Italy). Since only in two CAD patients 25OHD concentration was > of the cut off value of 30 ng/mL, sometimes used for optimal vitamin status, patients were classified according to 25OHD values reported by Thacher et al. [20]: 25OHD deficiency, 20 ng/mL. Statistical analysis Data are expressed as median and 25the75th percentiles or mean SD or number and percentage. The normality of data distribution was assessed by the KolmogoroveSmirnoff test. Fisher’s exact test was used for categorical outcomes. Differences across the three groups of 25OHD level were compared by one-way ANOVA followed by Tukey post-hoc test (for normally distributed parameters) or KruskaleWallis test followed by Dunn post-hoc test (for non-normally distributed parameters). Associations between parameters were examined by the Spearman correlation test. Data were analyzed using GraphPad Prism 5.0 biochemical statistical package (GraphPad Software, San Diego, CA). A p value 30 ng/mL. After patient stratification according to 25OHD level, the anthropometric parameters BMI, waist circumference, WHR as well as EAT
Reduced plasma 25OHD level is associated with increased VDR and reduced CYP27B1 and CYP24A1 expression at EAT level CAD patients were stratified according to plasma 25OHD level. Compared to 25OHD sufficient group, 25OHD deficient group displayed about a 2-fold increase in VDR expression (p < 0.05) (Fig. 1). A statistically significant reduction in CYP27B1 expression was observed in both 25OHD insufficient and deficient groups (p < 0.05 for both), as compared with 25OHD sufficient group (Fig. 1). CYP24A1 expression was also
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
4
E. Dozio et al.
Table 2 Demographic, anthropometric and biochemical characteristics of coronary artery disease patients classified according to plasma 25OHD level.
Age (years) Weight (Kg) BMI (kg/m2) Waist (cm) Hip (cm) WHR EAT thickness (mm) Fasting glucose (mg/dl) Fasting insulin (mU/ml) HOMA-IR Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) CRP (mg/dl) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) 25OHD (ng/mL)
25OHD 20 ng/mL
(n Z 24)
(n Z 21)
(n Z 9)
66.00, 58.25e72.50 74.00, 70.00e81.00 27.17, 25.13e28.63 104.00, 100.00e108.00 101.00, 91.75e106.00 1.02, 1.02e1.06 8.00, 6.25e9.25 82.50, 78.50e101.80 7.71, 5.08e13.51 1.50, 1.06e2.82 140.00, 129.00e167.00 34.00, 30.00e43.50 81.80, 70.85e109.00 117.00, 85.50e187.00 0.30, 0.20e1.00 120.00, 120.00e130.00 70.00, 70.00e70.00 6.88, 4.56e8.24a,b
66.00, 61.00e76.00 80.00, 69.00e90.50 27.75, 23.76e29.55 103.50, 92.50e114.00 105.00, 96.00e108.00 1.02, 0.99e1.06 9.00, 6.00e10.25 88.00, 75.25e116.30 6.75, 4.99e11.20 1.61, 1.07e2.61 138.50, 114.30e161.50 33.00, 25.50e42 77.20, 60.80e88.70 97.00, 75.50e143.00 0.45, 0.10e1.50 120.00, 112.50e127.50 70.00, 70.00e70.00 14.35, 11.64e16.74b
68.00, 58.50e75.00 78.00, 70.50e80.50 25.60, 24.43e28.74 102.00, 96.50e106.00 102.00, 96.50e107.00 1.02, 0.91e1.07 7.00, 6.00e8.50 80.00, 77.00e101.50 8.53, 4.91e13.43 2.16, 1.06e2.88 150.00, 117.50e167.50 44.00, 38.50e45.00 73.00, 58.20e104.10 104.00, 100e153.00 0.10, 1.10e1.20 130.00, 120.00e140.00 70.00, 70.00e80.00 23.70, 20.53e28.81
BMI: body mass index; CRP: C-reactive protein; EAT: epicardial adipose tissue; HOMA-IR: homeostatis model of insulin resistance; 25OHD: 25hydroxycholecalciferol; WHR: waist to hip ratio. Data are expressed as median and 25the75th percentiles. a p < 0.001 vs. 25OHD 10e20 ng/mL; b p < 0.001 vs. 25OHD >20 ng/mL (ANOVA).
decreased both in 25OHD deficient and insufficient groups (p < 0.05 and p < 0.01, respectively) (Fig. 1). Correlation analyses indicated that plasma 25OHD level was inversely correlated with VDR (r Z 0.48, p < 0.05) and positively with CYP27B1 (r Z 0.61, p < 0.001) and CYP24A1 (r Z 0.53, p < 0.05) expression at EAT level. Reduced plasma 25OHD level is associated to increased expression of inflammation-related molecules at EAT level CAD patients were stratified according to plasma 25OHD level. Compared to 25OHD sufficient group, 25OHD insufficient and deficient groups displayed about a 3-fold
increase in MCP-1 level (p < 0.05) (Fig. 2). A 2-fold increase in PTX-3 level has also been observed in the 25OHD deficient group, as compared with 25OHD sufficient group (p < 0.05) (Fig. 2). Expression of TNFa and IL-6 was up-regulated of about 2times in both insufficient and deficient groups, although these differences did not reach the statistical significance. Adiponectin level was almost the same in the three groups. Increased level of inflammation-related molecules is associated to increased VDR expression at EAT level CAD patients were further stratified according to tertiles (Q1-Q2-Q3) of VDR expression at EAT level.
Figure 1 Evaluation of VDR, CYP27B1 and CYP24A1 gene expression at EAT level according to plasma 25OHD concentration. CAD patients were stratified into three groups (sufficient, insufficient and deficient) according to plasma 25OHD concentration. Gene expression of VDR, CYP27B1 and CYP24A1 at EAT level is expressed as fold change SD vs. 25OHD sufficient group. Normalized raw data (mean SD) are also reported. *p < 0.05, p < 0.01 vs. 25OHD sufficient group.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
Epicardial adipose tissue inflammation is related to vitamin D deficiency
5
Figure 2 Gene expression of inflammation-related molecules at EAT level according to plasma 25OHD concentration. CAD patients were stratified into three groups (sufficient, insufficient and deficient) according to plasma 25OHD concentration. Gene expression of MCP-1, PTX3, TNFa, IL-6 and adiponectin at EAT level is expressed as fold change SD vs. 25OHD sufficient group. Normalized raw data (mean SD) are also reported. *p < 0.05 vs. 25OHD sufficient group.
Compared to Q1, MCP-1 and TNFa expression in Q2 was about 2.5-fold higher (p < 0.05, for both) (Fig. 3). We also observed about a 3-fold increase in MCP-1 (p < 0.05), a 2fold increase in PTX-3 (p < 0.05), a 2.5-fold increase in TNFa (p < 0.05) and a six-fold increase in IL-6 levels (p < 0.05) in Q3 compared to Q1 (Fig. 3). Adiponectin level was almost the same in the three groups. Correlation analyses also indicated a positive correlation between VDR and MCP-1 (r Z 0.69, p < 0.001), PTX-3 (r Z 0.45, p < 0.05), TNFa (r Z 0.49, p < 0.05) and IL-6 (r Z 0.66, p < 0.01) expression at EAT level, but not for adiponectin (r Z 0.27, p Z 0.24).
Discussion The novelty of our study is the observation that in CAD patients the expression of pro-inflammatory molecules at EAT level seems to be related to plasma 25OHD concentration and local expression of VDR. In fact, as 25OHD level decreases, local EAT expression of both VDR and proinflammatory cytokines increases. Moreover, patients with increased VDR expression also displayed higher level of pro-inflammatory mediators.
In addition to its well known role as a regulator of calcium homeostasis and bone metabolism, vitamin D may exert other different physiological functions. In particular, due to its ability to modulate T-lymphocyte proliferation and function and to suppress the production of inflammatory cytokines, vitamin D is recognized as a potential anti-inflammatory molecule [21e24]. Previous in vitro studies showed that vitamin D is able to down regulate the expression of pro-inflammatory mediators in human monocytes stimulated with interferon-g [11] and to inhibit the production of MCP-1 and other pro-inflammatory mediators in human preadipocytes [13] and mature adipocytes [14]. Although these previous studies suggested that vitamin D may protect against adipose tissue inflammation by disrupting the deleterious cycle of macrophage recruitment, our study represent the first human study on the potential association between vitamin D and inflammation at EAT level in CAD patients. As previously observed for other adipose depots, i.e. subcutaneous and visceral [25], also EAT expresses the activating 1ahydroxylase enzyme CYP27B1, the degrading 24hydroxylase enzyme CYP24A1 and VDR, thus suggesting the existence of a paracrineeautocrine effect of vitamin D at EAT level.
Please cite this article in press as: Dozio E, et al., Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/ j.numecd.2014.08.012
6
E. Dozio et al.
Figure 3 Gene expression of inflammation-related molecules at EAT level according to tertiles of VDR expression. CAD patients stratified according to tertiles (Q1-Q2-Q3) of VDR expression at EAT level. Gene expression of MCP-1, PTX3, TNFa, IL-6 and adiponectin at EAT level is expressed as fold change SD vs. Q1. Normalized raw data (mean SD) are also reported. *p < 0.05.
According to previous reports, our data seem to suggest that vitamin D may be involved in the regulation of inflammation also at EAT. In fact, in condition of plasma 25OHD insufficiency and deficiency, the local vitamin D metabolism seems to be suppressed and associated to an increased production of inflammatory mediators. We have also observed an increased expression of VDR associated both to reduced plasma 25OHD level as well as to increased EAT inflammation. Recent studies suggested that VDR expression may be up-regulated in adipocytes after exposure to macrophages-derived products [26] and in visceral fat VDR level was higher in obese than lean women [25]. Our data do not directly prove the molecular mechanisms inducing VDR up-regulation, anyway, given the potential anti-inflammatory action of vitamin D, this could be a counter-regulatory response to ameliorate inflammation. The open question is whether vitamin D supplementation could be a useful tool to counteract local EAT inflammation in these patients. The prevalence of hypovitaminosis D (
