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Research Article
Originally Published 24 March 2016
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Local Production of Fatty Acid–Binding Protein 4 in Epicardial/Perivascular Fat and Macrophages Is Linked to Coronary Atherosclerosis

Masato Furuhashi, Takahiro Fuseya, Masaki Murata, Kyoko Hoshina, Shutaro Ishimura, Tomohiro Mita, Yuki Watanabe, … Show All … , Akina Omori, Megumi Matsumoto, Takeshi Sugaya, Tsuyoshi Oikawa, Junichi Nishida, Nobuaki Kokubu, Marenao Tanaka, Norihito Moniwa, Hideaki Yoshida, Norimasa Sawada, Kazuaki Shimamoto, and Tetsuji MiuraAuthor Info & Affiliations
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume 36, Number 5
https://doi.org/10.1161/ATVBAHA.116.307225
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Abstract

Objective—

Fatty acid–binding protein 4 (FABP4) is expressed in adipocytes and macrophages, and elevated circulating FABP4 level is associated with obesity-mediated metabolic phenotype. We systematically investigated roles of FABP4 in the development of coronary artery atherosclerosis.

Approach and Results—

First, by immunohistochemical analyses, we found that FABP4 was expressed in macrophages within coronary atherosclerotic plaques and epicardial/perivascular fat in autopsy cases and macrophages within thrombi covering ruptured coronary plaques in thrombectomy samples from patients with acute myocardial infarction. Second, we confirmed that FABP4 was secreted from macrophages and adipocytes cultured in vitro. Third, we investigated the effect of exogenous FABP4 on macrophages and human coronary artery–derived smooth muscle cells and endothelial cells in vitro. Treatment of the cells with recombinant FABP4 significantly increased gene expression of inflammatory markers in a dose-dependent manner. Finally, we measured serum FABP4 level in the aortic root (Ao-FABP4) and coronary sinus (CS-FABP4) of 34 patients with suspected or known coronary artery disease. Coronary stenosis score assessed by the modified Gensini score was weakly correlated with CS-FABP4 but was not correlated with Ao-FABP4. A stronger correlation (r=0.59, P<0.01) was observed for the relationship between coronary stenosis score and coronary veno-arterial difference in FABP4 level, (CS-Ao)-FABP4, indicating local production of FABP4 during coronary circulation in the heart. Multivariate analysis indicated that (CS-Ao)-FABP4 was an independent predictor of the severity of coronary stenosis after adjustment of conventional risk factors.

Conclusions—

FABP4 locally produced by epicardial/perivascular fat and macrophages in vascular plaques contributes to the development of coronary atherosclerosis.

Introduction

Fatty acid–binding proteins (FABPs) are ≈14- to 15-kDa predominantly cytosolic proteins that can reversibly bind to saturated and unsaturated long-chain fatty acids with high affinity.1–3 It has been proposed that FABPs facilitate the transport of lipids to specific compartments in the cell. FABP4, known as adipocyte FABP (A-FABP) or aP2, is expressed in both adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis.4–7 We previously proposed that inhibition of FABP4 in cells would be a novel therapeutic strategy against insulin resistance, diabetes mellitus, and atherosclerosis.8
FABP4 has been shown to be secreted from adipocytes in association with lipolysis via a nonclassical secretion pathway,9–11 though there are no typical secretory signal peptides in the sequence of FABP4.1 Recent studies have shown that FABP4 might act as an adipokine, an adipocyte-derived bioactive molecule, for the development of insulin resistance in the liver10 and suppression of cardiomyocyte contraction.12 It has also been shown that elevation of the circulating FABP4 level is associated with obesity, insulin resistance, hypertension, cardiac diastolic dysfunction, and atherosclerosis.9,13–18 Notably, increased plasma level of FABP4 was shown to be independently associated with the presence of coronary artery disease.19 Moreover, FABP4 was found in human atherosclerotic plaques, and its presence was associated with high-risk phenotypes of atherosclerotic plaques, that is, features of inflammatory and vulnerable plaques.20,21
Much attention is currently focused on roles of epicardial/perivascular adipose tissue in the development of cardiovascular diseases. It has recently been proposed that epicardial/perivascular fat promotes cardiovascular disease through secretion of several adipokines.22 However, little is known about the roles of FABP4 locally produced in the heart: the origin of FABP4 from epicardial/perivascular fat, the level of FABP4 released into coronary circulation and the impact of secreted FABP4 on development of atherosclerosis.
We hypothesized that FABP4 locally produced by epicardial/perivascular fat and macrophages of vascular plaques contributes to the development of coronary atherosclerosis. To test this hypothesis, we systematically examined the localization of FABP4 in atherosclerotic plaques and around fat of the coronary artery, FABP4 release from macrophages and adipocytes, FABP4-induced gene expression in several cell types, and the level of FABP4 in coronary circulation and its correlation with severity of coronary stenosis. The results of this present study supported our hypothesis.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Detection of FABP4 Expression in the Coronary Artery and Epicardial/Perivascular Fat

Representative hematoxylin and eosin staining of the coronary artery in an autopsy case with ischemic heart disease (male; 68 years old) is shown in Figure 1A. Immunohistological staining using anti-CD68 and anti-FABP4 antibodies showed that FABP4 was present in some, but not all, macrophages of coronary atherosclerotic lesions (Figure 1B). In epicardial/perivascular fat, FABP4 was detected in both adipocytes and macrophages (Figure 1C). FABP4 in the atherosclerotic lesions and that in epicardial/pericardial fat were similarly observed in the other 19 autopsy cases.
Figure 1. Fatty acid–binding protein 4 (FABP4) expression in coronary atherosclerotic plaque, epicardial/perivascular fat, and coronary thrombus. A, Representative hematoxylin and eosin (H–E) staining in atherosclerotic plaques of the coronary artery and epicardial/perivascular fat in an autopsy case with ischemic heart disease (male; 68 years old). Square boxes in A are magnified in B and C. B and C, Immunohistological staining with CD68 (brown) or FABP4 (brown) and double immunohistological staining with CD68 (blue) and FABP4 (brown) in atherosclerotic plaques of the coronary artery (B) and epicardial/perivascular fat (C). Red arrows indicate FABP4-positive macrophages. D and E, Representative H–E staining and immunohistological staining with CD68 (brown) or FABP4 (brown, D) and double immunofluorescence staining with CD68 (green) and FABP4 (red, E) in coronary thrombi obtained by thrombectomy in patients with acute myocardial infarction. Images of a wide area (D) were reconstructed using a BIOREVO BZ-9000 with a BZ-II analyzer. Scale bars indicate 1.0 mm (A), 100 µm (B and C), 500 µm (D), and 20 µm (E).

Detection of FABP4 Expression in Coronary Arterial Thrombectomy Specimens

Coronary thrombectomy specimens were obtained from 32 patients with acute myocardial infarction, and the specimens were histologically classified into 2 major types: thrombi only and thrombi containing atherosclerotic plaque components. As shown in Figure 1D, some of thrombus specimens contained atherosclerotic plaque components, including macrophages immunohistologically stained by anti-CD68 antibody and cholesterol clefts. FABP4-expressing cells were present in the atherosclerotic component admixed with the coronary arterial thrombus (Figure 1D). To determine whether FABP4 is expressed in macrophages, double immunofluorescence analysis using anti-CD68 (green) and anti-FABP4 (red) antibodies was carried out. Immunofluorescence staining showed colocalization (yellow) of FABP4 in some, but not all, macrophages within thrombi containing atherosclerotic plaque (Figure 1E).

Secretion of FABP4 From Adipocytes and Macrophages

Western blot analysis showed that FABP4 was present in both the cell lysate and the conditioned medium of 3T3-L1 adipocytes and that glyceraldehyde 3-phosphate dehydrogenase, a nonsecretory protein, was not present in the conditioned medium (Figure 2A), indicating that FABP4 in the conditioned medium was a result of its secretion from 3T3-L1 adipocytes, not a result of its leakage via injured cell membranes as previously reported.10,11 FABP4 was detected in phorbol 12-myristate 13-acetate–stimulated human THP-1 macrophages, but not in nonstimulated THP-1 monocytic cells, in the cell lysate (Figure 2B) as previously reported.5,8 FABP4 was secreted from several macrophage cell lines, including phorbol 12-myristate 13-acetate–stimulated THP-1 cells (Figure 2B), primary mouse macrophages (Figure 2C), mouse J774.1 cells (Figure 2D), and mouse RAW264.7 cells (Figure 2E).
Figure 2. Secretion and actions of fatty acid–binding protein 4 (FABP4) in macrophages. A–D, Western blot analysis of FABP4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the cell lysate (CL) and conditioned medium (CM) for 24 hours of mouse differentiated 3T3-L1 adipocytes (A), human monocytic THP-1 cells treated with and without 100-nmol/L phorbol 12-myristate 13-acetate (PMA, B), mouse peritoneal primary macrophages (C), mouse J774.1 macrophages (D), and mouse RAW264.7 macrophages (E). F, Microarray analysis in RAW264.7 macrophages treated with 200-nmol/L recombinant FABP4 in the absence and presence of 0.2-mmol/L palmitic acid (PA) for 24 hours. The Venn diagram shows the number of genes significantly (P<0.05) regulated by exogenous FABP4 with and without PA. G–I, Gene expression of inflammatory markers, including Mcp1 (G), Il6 (H), and Tnfa (I), determined by quantitative real-time polymerase chain reaction in RAW264.7 macrophages treated with 0- to 200- nmol/L recombinant FABP4 in the medium supplemented with 0.5% fatty acid–free bovine serum albumin in the absence and presence of 0.2-mmol/L linoleic acid (LA) or 0.2-mmol/L PA for 24 hours. *P<0.05 vs FABP4 (0 nmol/L)-(−). †P<0.05 vs FABP4 (0 nmol/L)-PA (+).

Fatty Acid–Binding Affinity for FABP4

Fatty acid–binding affinity for recombinant FABP4 at a basal state and under an oxidative condition induced by 2,2′-azobis(2-amidinopropane) dihydrochloride, a free radical generator, is shown in the Table. The Kd values of 4 abundant long-chain fatty acids in human blood,23 that is, palmitic acid, stearic acid, oleic acid, and linoleic acid, were 11.7±1.9, 10.2±1.0, 7.7±1.0, and 1.0±0.2 µmol/L, respectively, indicating that linoleic acid, an essential polyunsaturated fatty acid, had the highest affinity for FABP4 under a basal condition. The Kd values of fatty acids for bovine serum albumin were higher than those for FABP4, showing that FABP4 generally had higher affinity and selectivity for long-chain fatty acids than did albumin. On exposure to 2,2′-azobis(2-amidinopropane) dihydrochloride, the ratio of Kd for FABP4 was significantly increased in most of the tested fatty acids except for palmitic acid (Table). The results indicate that palmitic acid, a saturated fatty acid, came to have relatively higher affinity for FABP4 under a specific condition, such as obesity-induced oxidative stress.
Table. Fatty Acid-Binding Affinity Assay Using 1,8-ANS
Fatty acidC:DKd for FABP4Kd for BSA(µmol/L)
Basal (µmol/L)+AAPH(fold change)
Palmitic acidC16:011.7±1.9*1.010.7±2.0
Palmitoleic acidC16:12.4±0.57.5†1.1±0.6
Stearic acidC18:010.2±1.0*1.4†15.0±2.1
Oleic acidC18:17.7±1.0*4.0†8.3±1.2
Linoleic acidC18:21.0±0.22.5†10.0±2.2
α-Linolenic acidC18:3ω31.9±0.31.8†8.7±1.3
Arachidonic acidC20:44.0±0.9*3.8†9.2±0.9
Eicosapentaenoic acidC20:56.7±1.0*3.6†21.6±3.4
Docosahexaenoic acidC22:63.4±0.7*1.7†12.7±2.5
Variables are expressed as means or means±SEM. 1,8-ANS indicates 1-anilinonapthalene 8-sulfonic acid; AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; BSA, bovine serum albumin; and FABP4, fatty acid–binding protein 4.
*
P<0.05 vs linoleic acid.
†
P<0.05 vs basal.

Effects of Exogenous FABP4 in RAW264.7 Macrophages

Microarray analysis of gene expression was performed in RAW264.7 macrophages treated with 200-nmol/L recombinant FABP4 in the absence and presence of 0.2-mmol/L palmitic acid. After quality control by removing low signal and flagged genes and control spots, a total of 32 079 genes were analyzed among the 4 groups (Figure IA in the online-only Data Supplement). Recombinant FABP4 and palmitic acid significantly affected the expression of 288 genes (Figure IB and IC in the online-only Data Supplement). In the absence and presence of palmitic acid, 1308 of 29 298 (Figure ID and IE in the online-only Data Supplement) and 1631 of 28 687 (Figure IF and IG in the online-only Data Supplement) quality-controlled genes, respectively, were significantly regulated by treatment with exogenous FABP4. As shown in Figure 2F, the expression of 1214 and 1537 independent genes was significantly changed by treatment with recombinant FABP4 in the absence and presence of palmitic acid, respectively. There were 94 common genes that were significantly regulated by treatment with FABP4 regardless of the presence of palmitic acid. Interestingly, some of the common genes were regulated by palmitic acid in the same direction, but the others were regulated in a different direction (Figure IIA–IID in the online-only Data Supplement). Results of gene ontology analysis for exogenous FABP4 treatment with and without palmitic acid are shown in Table II in the online-only Data Supplement. Enriched gene ontology categories were regulations of biological process, cellular process, metabolic process, cell proliferation, and developmental process. Pathway analysis showed that there were several FABP4-regulated pathways, which depended on the absence or presence of palmitic acid (Table III in the online-only Data Supplement). Notably, inflammation-related pathways, including the chemokine signaling pathway, tumor necrosis factor α and nuclear factor-κB signaling pathway, and toll-like receptor (TLR) signaling pathway, were significantly regulated by treatment with FABP4 in the presence, but not in the absence, of palmitic acid.
To address whether FABP4 has distinct effects in a ligand-dependent manner, gene expression of inflammatory markers was examined in mouse RAW264.7 macrophages treated with recombinant FABP4 (0–200 nmol/L) in the absence and presence of ligands for FABP4, including an essential polyunsaturated fatty acid, linoleic acid, as the ligand with highest affinity for FABP4 and a saturated fatty acid, palmitic acid, as a ligand with relatively high affinity for FABP4 under an oxidative condition (Table). Gene expression of monocyte chemotactic protein-1 was unchanged by FABP4 treatment in the presence or absence of linoleic acid. However, FABP4 treatment significantly augmented the increase in monocyte chemotactic protein-1 gene expression by palmitic acid in a dose-dependent manner (Figure 2G). Similar results of the ligand-dependent effect of FABP4 were obtained for gene expression of interleukin-6 (IL-6; Figure 2H) and tumor necrosis factor α (Figure 2I).

Effects of Exogenous FABP4 in Human Coronary Artery Smooth Muscle Cells

Treatment of human coronary artery smooth muscle cells with recombinant FABP4 in the presence of palmitic acid significantly increased the gene expression of inflammatory cytokines, including monocyte chemotactic protein-1 (Figure 3A), IL-6 (Figure 3B), and tumor necrosis factor α (Figure 3C), in a dose-dependent manner. However, there was no such an effect in the absence of palmitic acid. However, recombinant FABP4 increased the expression of proliferative phenotype–related genes, including platelet-derived growth factor receptor α (Figure 3D), platelet-derived growth factor receptor β (Figure 3E), and myosin heavy chain 10 (Figure 3F), in a dose-dependent manner regardless of the presence of palmitic acid. MTS and bromodeoxyuridine assays showed that treatment of human coronary artery smooth muscle cells with recombinant FABP4 significantly increased cell proliferation in a dose-dependent manner (Figure 3G and 3H). The scratch wound-healing assay showed that FABP4-treated human coronary artery smooth muscle cells migrated faster than did untreated cells (Figure 3I).
Figure 3. Effect of exogenous fatty acid–binding protein 4 (FABP4) in human coronary artery smooth muscle cells. A–F, Gene expression of inflammatory markers, including Mcp1 (A), Il6 (B), and Tnfa (C), and proliferative phenotype-related markers, including Pdgfra (D), Pdgfrb (E), and Myh10 (F), determined by quantitative real-time polymerase chain reaction in human coronary artery smooth muscle cells (HCASMC) treated with recombinant FABP4 (0–200 nmol/L) in the absence and presence of 0.2-mmol/L palmitic acid (PA) for 24 hours. *P<0.05 vs FABP4 (0 nmol/L)-PA (−). †P<0.05 vs FABP4 (0 nmol/L)-PA(+). G and H, Proliferation of HCASMC with 0- to 200- nmol/L recombinant FABP4 in the supplement of 0.1% FBS for 24 hours assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; G) and bromodeoxyuridine (BrdU, H) assays. *P<0.05 vs FABP4 (0 nmol/L). I, Migration of HCASMC with 0- to 200- nmol/L recombinant FABP4 for 15 hours in the supplement of 0.5% bovine serum albumin (BSA) assessed by scratch wound assay. *P<0.05 vs FABP4 (0 nmol/L).

Effects of Exogenous FABP4 in Human Vascular Endothelial Cells

Treatment of human coronary artery endothelial cells with FABP4 in the presence of palmitic acid significantly increased the gene expression of inflammatory cytokines, including monocyte chemotactic protein-1 (Figure 4A), IL-6 (Figure 4B), and tumor necrosis factor α (Figure 4C), in a dose-dependent manner, but there was no such an effect in the absence of palmitic acid. Similar results were obtained in human umbilical vein endothelial cells, another cell line of vascular endothelial cells (Figure 4D–4F). Furthermore, treatment with recombinant FABP4 significantly decreased phosphorylation of endothelial nitric oxide synthase regardless of the presence of palmitic acid in both human coronary artery endothelial cells (Figure 4G) and human umbilical vein endothelial cells (Figure 4H).
Figure 4. Effect of exogenous fatty acid–binding protein 4 (FABP4) in human vascular endothelial cells. A–C, Gene expression of inflammatory markers, including Mcp1 (A), Il6 (B), and Tnfa (C), determined by quantitative real-time polymerase chain reaction (PCR) in human coronary artery endothelial cells (HCAEC) treated with 0- to 200-nmol/L recombinant FABP4 in the absence and presence of 0.2 mmol/L palmitic acid (PA) for 24 hours. D–F, Gene expression of inflammatory markers, including Mcp1 (D), Il6 (E), and Tnfa (F), determined by quantitative real-time PCR in human umbilical vein endothelial cells (HUVEC) treated with 0- to 200-nmol/L recombinant FABP4 in the absence and presence of 0.2-mmol/L PA for 24 hours. *P<0.05 vs FABP4 (0 nmol/L)-PA (−). †P<0.05 vs FABP4 (0 nmol/L)-PA (+). G and H, Western blot analysis of phospho-endothelial nitric oxide synthase (eNOS; pS1177), eNOS, tubulin, and vinculin in treatment of HCAEC (G) and HUVEC (H) with 0- to 200-nmol/L recombinant FABP4 in the absence and presence of 0.2-mmol/L PA for 6 and 9 hours, respectively. The graphs to the right of the blots show the quantification from experiments performed. *P<0.05.

Intracardiac Veno-Arterial Difference in FABP4 and Coronary Stenosis

Clinical characteristics of the 34 enrolled patients are shown in Table IV in the online-only Data Supplement. Mean age, body mass index, and waist circumference were 64.9±1.8 years, 24.5±0.3 kg/m2, and 87.7±1.0 cm, respectively. Patients had diabetes mellitus (50.0%), dyslipidemia (52.9%), and hypertension (64.7%), and most of the patients had received antihypertensive agents and statins. Serum FABP4 levels in the vein (V-FABP4), aortic root (Ao-FABP4), and coronary sinus (CS-FABP4) were 18.1±1.5, 14.2±1.3, and 17.1±1.5 ng/mL, respectively (Table IV in the online-only Data Supplement). CS-FABP4 level was significantly higher than Ao-FABP4 level. In a simple regression analysis, levels of V-FABP4, Ao-FABP4, and CS-FABP4 were negatively correlated with estimated glomerular filtration rate and were positively correlated with age, body mass index, waist circumference and levels of glucose, insulin, homeostasis model assessment of insulin resistance, and high-sensitivity C-reactive protein (Table V in the online-only Data Supplement).
Coronary stenosis score was weakly correlated with logarithmically transformed V-FABP4 (r=0.366, P=0.033) and CS-FABP4 (r=0.414, P=0.015) but was not correlated with logarithmically transformed Ao-FABP4 (r=0.305, P=0.080; Figure 5A–5C) or other variables (Table VI in the online-only Data Supplement). A stronger correlation (r=0.592, P<0.001) was observed for the relationship between coronary stenosis score and coronary veno-arterial difference in FABP4 level, (CS-Ao)-FABP4, an index of FABP4 production in the heart (Figure 5D). Multivariate analyses adjusted by conventional coronary risk factors, including age, body mass index, smoking and the presence of diabetes mellitus, dyslipidemia, and hypertension, indicated that (CS-Ao)-FABP4 was an independent predictor of the severity of coronary stenosis, explaining a total of 67% of the variance in this measure (R2=0.67; Table VII in the online-only Data Supplement).
Figure 5. Circulating fatty acid–binding protein 4 (FABP4) levels and coronary atherosclerosis. A–C, Coronary stenosis assessed by the modified Gensini method was plotted against logarithmically transformed FABP4 level in the vein (log V-FABP4, A), aorta sinus (log Ao-FABP4, B) and coronary sinus (log CS-FABP4, C) for each subject. D, Coronary stenosis assessed by the modified Gensini method was plotted against coronary veno-arterial difference in FABP4 level, (CS-Ao)-FABP4, an index of FABP4 production during coronary circulation in the heart, for each subject.

Discussion

This study showed the expression of FABP4 in epicardial/perivascular fat and in macrophages within atherosclerotic lesions in humans and showed the secretion of FABP4 from both adipocytes and macrophages in vitro. This study also showed for the first time that serum FABP4 level was significantly higher in the CS than in the Ao in patients who underwent cardiac catheterization, suggesting that FABP4 is released into the coronary circulation in the heart. In addition, a strongly positive correlation was observed between the transcardiac gradient of FABP4 in blood, (CS-Ao)-FABP4, and the stenosis score in the coronary artery, indicating that locally produced FABP4 contributes to the development of coronary atherosclerosis. Furthermore, in vitro experiments indicated that exogenous FABP4, which may be locally produced in epicardial/perivascular fat and macrophages within the atherosclerotic lesion, acts in several vascular cells, including macrophages, vascular smooth muscle cells, and vascular endothelial cells, leading to the development of coronary atherosclerosis by cooperating with accelerating vascular inflammation, proliferation, and migration of smooth muscle cells and impaired endothelial function. Thus, the present findings collectively support the hypothesis that FABP4 derived from epicardial/perivascular adipocytes and macrophages localized in the artery significantly contributes to the progression of coronary atherosclerosis (Figure 6).
Figure 6. Possible mechanisms of the development of coronary atherosclerosis by locally produced fatty acid–binding protein 4 (FABP4). Secreted FABP4, which is locally produced in epicardial/perivascular fat and macrophages in the atherosclerotic lesion, acts as a biological molecule in several vascular cells, including macrophages, smooth muscle cells (SMC), and endothelial cells (EC), in a paracrine and autocrine manner. Local production of FABP4 leads to the development of coronary atherosclerosis by cooperating with accelerating vascular inflammation, proliferation and migration of smooth muscle cells, and impaired endothelial function.
Epicardial fat shares a common embryological origin with mesenteric and omental fat, and the origin is the splanchnopleuric mesoderm associated with the gut.24 Compared with recent reports about association of visceral fat with metabolic syndrome and atherosclerosis, little attention had been paid to the roles of epicardial adipose tissue, which surrounds the outer layer of coronary artery vessels. However, it has recently been demonstrated that epicardial/perivascular adipose tissue secretes several adipokines to regulate vessels in a paracrine manner, potentially accelerating the development of cardiovascular disease.25 Epicardial fat may directly influence coronary atherogenesis and myocardial function because there is no fibrous fascial layer to impede diffusion of free fatty acids and adipokines from the fat underlying the myocardium and vessel.22 FABP4 level in the local atherosclerotic area should be much higher than that in circulating blood (≈1 nmol/L; 15 ng/mL), and we therefore used recombinant FABP4 at the dose of 0 to 200 nmol/L in in vitro experiments in this study. Interestingly, segments of coronary arteries lacking epicardial fat or separated from it by a bridge of myocardial tissue have been reported to be protected against the development of atherosclerosis.26 Consequently, FABP4 expression found in epicardial/perivascular fat and macrophages within the atherosclerotic lesion and secretion of FABP4 from both adipocytes and macrophages (Figures 1 and 2) support the hypothesis of direct paracrine/autocrine effects of FABP4 as a bioactive molecule on the development of coronary atherosclerosis.
Systemic fat stores are the principal source of free fatty acids for the heart.27 Free fatty acid kinetic studies showed that under normal basal conditions, endogenous free fatty acids, which are thought to be derived from lipolysis in epicardial adipose tissue,28 were released into the coronary veins and then into the coronary venous sinus.27,28 It has been demonstrated that epicardial fat has ≈2-fold higher rates of lipolysis and lipogenesis than those of other fat depots in guinea pig.29,30Notably, FABP4 mRNA expression in epicardial adipose tissue has been reported to be profoundly increased compared with its expression in paraaortic adipose tissue in patients with metabolic syndrome.31 Sympathetic nerve activation and several inflammatory cytokines are known to increase lipolysis in adipocytes.32 Moreover, FABP4 is secreted from adipocytes under regulation by the lipolytic signal pathway, though FABP4 lacks an N-terminal secretory signal sequence.1,3,10,11 Therefore, the increase of FABP4 production during coronary circulation in patients with coronary stenosis may be, at least in part, because of increase in lipolysis via activation of sympathetic nerve tone and inflammatory cytokines. Increased atherosclerotic lesion and its attendant inflammation also may lead to increased secretion of FABP4.
This study showed that FABP4 was expressed in some, but not all, macrophages in coronary plaques. The heterogeneous expression of FABP4 may reflect differences in level and stimuli of macrophage activation. It has been shown that activation of macrophages by several inflammatory stimuli, including lipopolysaccharide, phorbol 12-myristate 13-acetate, and oxidized low-density lipoprotein, induces expression of FABP4 in macrophages.5,33,34 Expression of FABP4 has been shown to be significantly lower in macrophages than in adipocytes.5,35 However, it is possible that the level of FABP4 secreted from macrophages is sufficient for causing significant effects on nearby smooth muscle and endothelial cells, leading to an inflammatory response, proliferation, and direct migration from the media into the intima.
Microarray analysis showed that treatment of RAW264.7 macrophages with exogenous FABP4 was significantly associated with inflammation-related pathways in the presence, but not in the absence, of palmitic acid (Table III in the online-only Data Supplement). In fact, induction of inflammatory genes, including Mcp1, Il6, and Tnfa, by exogenous FABP4 in RAW264.7 macrophages was dependent on palmitic acid, but not linoleic acid, as a ligand (Figure 2G–2I). In fatty acid–binding assays, linoleic acid had the highest affinity for FABP4 under a basal condition (Table), suggesting that FABP4 usually acts as a carrier of linoleic acid, an essential fatty acid. However, palmitic acid, a saturated fatty acid, had relatively higher affinity for FABP4 under oxidative stress condition. This finding is consistent with the results of a previous study showing that the affinity of FABP4 for fatty acids was reduced by treatment with 4-hydroxy-2-nonenal.36 Similar findings about relative alteration of the affinity were reported for another FABP, liver FABP (FABP1), through conformation change of structure.37,38 It has been shown that obesity and metabolic syndrome cause oxidative stress in adipose tissue.39 Thus, under such a condition, FABP4 secreted from the adipose tissue might be conformationally changed to increase binding to palmitic acid rather than to linoleic acid, resulting in augmented inflammatory responses. It has been shown that palmitic acid activates the TLR4 signaling pathway.40 Pathway analysis using microarray data showed that the TLR signaling pathway was regulated by treatment with FABP4 in the presence, but not in the absence, of palmitic acid (Table III in the online-only Data Supplement). The molecular mechanism underlying the induction of inflammatory genes by FABP4 in the presence of palmitic acid might be related to interaction between FABP4 and TLR4.
The present experiments also demonstrated that recombinant FABP4 induced dose-dependent proliferation and migration of human coronary artery smooth muscle cells and decreased endothelial nitric oxide synthase activation in human coronary artery endothelial cells and human umbilical vein endothelial cells, findings that are consistent with previous reports.41,42 Interestingly, those effects were ligand-independent unlike the effect of FABP4 on inflammatory responses. The difference in ligand selectivity between the outcomes of FABP4 treatment suggests that there are distinct regulatory mechanisms for multiple functions of FABP4. Nevertheless, an excess of local production of FABP4 might lead to the development of atherosclerosis.
Evidence indicating that FABP4 acts as a biological molecule is accumulating,10,12,41–43 and serum FABP4 level has been reported to predict long-term cardiovascular events.14,17,44 However, the receptor for FABP4 remains obscure. Furthermore, it is not known whether extracellular FABP4 is internalized into the cell or whether it acts by an intracellular signaling mechanism. A further understanding of the mechanism of FABP4 action may enable the development of new therapeutic strategies for cardiovascular and metabolic diseases, such as neutralization of FABP4 and blockade of the FABP4 receptor, if any.
This study has some limitations. First, most of the patients in the clinical study had been treated with drugs, including angiotensin II receptor blockers and statins, which have been reported to modulate FABP4 concentration.45–48 Therefore, serum FABP4 data might have been modified by such drugs. Second, the number of patients in the clinical study was small, and the possibility of a type I or II error in statistical tests cannot be excluded. Prospective studies using a larger number of subjects are needed to determine whether local production of FABP4 is indeed a major determinant of subsequent development of atherosclerosis. Furthermore, it has been shown that FABP4 concentration is sex related, being higher in females than in males, probably because of difference in adiposity.9,16 Although we enrolled only male subjects in this study to adjust for confounding factors, whether the present findings are applicable to female subjects remains to be examined. Finally, the veno-arterial transcardiac gradient of FABP4 level is indirect evidence of the production of FABP4 in the coronary artery. However, we cannot rule out the possibility that significant stenosis and previous infarct areas might reduce coronary flow and perfusion. Inability to measure the total amount of FABP4 production because the CS flow was not measured is a limitation of this study. Further studies are needed to verify the relationship between locally produced FABP4 and an atherosclerotic lesion in the coronary artery.
In conclusion, our findings support the notion that FABP4 locally produced by epicardial/perivascular fat and macrophages within vascular plaques contributes to the development of coronary atherosclerosis. A further understanding of local production of FABP4 from adipocytes and macrophages may enable the development of new therapeutic strategies for cardiovascular and metabolic diseases.

Acknowledgments

We thank Yoko Tamura for experimental management and technical help.

Significance

Fatty acid–binding protein 4 (FABP4) is expressed in macrophages within coronary atherosclerotic plaques and epicardial/perivascular fat in autopsy cases and macrophages within thrombi covering ruptured coronary plaques in thrombectomy samples from patients with acute myocardial infarction. FABP4 is secreted from macrophages in similar to adipocytes. Exogenous FABP4 acts in several vascular cells, including macrophages, vascular smooth muscle cells, and vascular endothelial cells, leading to the development of coronary atherosclerosis by cooperating with accelerating vascular inflammation, proliferation, and migration of smooth muscle cells and impaired endothelial function. Coronary stenosis score is strongly correlated with coronary veno-arterial difference in FABP4 level, indicating local production of FABP4 in the heart. FABP4 locally produced by epicardial/perivascular fat and macrophages in vascular plaques contributes to the development of coronary atherosclerosis.

Footnote

Nonstandard Abbreviations and Acronyms

Ao
aortic root
CS
coronary sinus
FABP4
fatty acid–binding protein 4
IL-6
interleukin-6

Supplemental Material

File (atvb_atvb-2016-307225_supp1.pdf)
File (atvb_atvb-2016-307225_supp2.pdf)

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Shown is a cross-section of a Drebrin haploinsufficient mouse carotid artery 4 weeks post endothelial denudation. An actin-binding protein that is upregulated in atherosclerosis and in response to endothelial injury, Drebrin inhibits smooth muscle cell migration and proliferation through stabilization of actin filaments. (See pages 984–993.)

Arteriosclerosis, Thrombosis, and Vascular Biology
Volume 36 • Number 5 • May 2016
Pages: 825 - 834
PubMed: 27013610

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© 2016 American Heart Association, Inc.

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Received: 19 January 2016
Accepted: 14 March 2016
Published online: 24 March 2016
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Keywords

  1. adipokine
  2. atherosclerosis
  3. coronary stenosis
  4. fatty acid–binding protein 4
  5. macrophage

Subjects

  1. Atherosclerosis
  2. Coronary Artery Disease
  3. Lipids and Cholesterol
  4. Stenosis
  5. Vascular Biology

Authors

Affiliations

Masato Furuhashi
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Takahiro Fuseya
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Masaki Murata
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Kyoko Hoshina
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Shutaro Ishimura
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Tomohiro Mita
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Yuki Watanabe
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Akina Omori
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Megumi Matsumoto
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Takeshi Sugaya
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Tsuyoshi Oikawa
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Nobuaki Kokubu
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Marenao Tanaka
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Norihito Moniwa
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Kazuaki Shimamoto
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Tetsuji Miura
From the Departments of Cardiovascular, Renal, and Metabolic Medicine (M.F., T.F., K.H., S.I., T.M., Y.W., A.O., M.M., J.N., N.K., M.T., N.M., H.Y., T.M.) and Molecular and Cellular Pathology (M.M., N.S.), Sapporo Medical University School of Medicine, Sapporo, Japan; Sapporo Medical University, Chuo-ku, Sapporo, Japan (K.S.); Department of Cardiovascular Internal Medicine, Obihiro Kosei Hospital, Obihiro, Japan (S.I., T.M.); Department of Nephrology and Hypertension, St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki, Kanagawa, Japan (T.S.); and CIMIC Co, Ltd, Yushima, Bunkyo-ku, Tokyo, Japan (T.S., T.O.).
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Notes

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.116.307225/-/DC1.
Correspondence to Masato Furuhashi, MD, PhD, Department of Cardiovascular, Renal, and Metabolic Medicine, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan. E-mail [email protected]

Disclosures

None.

Sources of Funding

M. Furuhashi has been supported by grants from JSPS KAKENHI, MEXT Translational Research Network Program, Uehara Memorial Foundation, SENSHIN Medical Research Foundation, Japan Diabetes Foundation, Takeda Medical Research Foundation, Ono Medical Research Foundation, Takeda Science Foundation, Akiyama Life Science Foundation, Yamaguchi Endocrine Research Foundation, Naito Foundation Natural Science Scholarship, Suhara Memorial Foundation, and Kondou Kinen Medical Foundation.

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Local Production of Fatty Acid–Binding Protein 4 in Epicardial/Perivascular Fat and Macrophages Is Linked to Coronary Atherosclerosis
Arteriosclerosis, Thrombosis, and Vascular Biology
  • Vol. 36
  • No. 5

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Arteriosclerosis, Thrombosis, and Vascular Biology
  • Vol. 36
  • No. 5
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