Local Production of Fatty Acid–Binding Protein 4 in Epicardial/Perivascular Fat and Macrophages Is Linked to Coronary Atherosclerosis
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.
FABP4 locally produced by epicardial/perivascular fat and macrophages in vascular plaques contributes to the development of coronary atherosclerosis.
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.
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.
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).
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.
|Fatty acid||C:D||Kd for FABP4||Kd for BSA(µmol/L)|
|Basal (µmol/L)||+AAPH(fold change)|
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).
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).
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).
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).
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.
fatty acid–binding protein 4
We thank Yoko Tamura for experimental management and technical help.
Sources of Funding
M. Furuhashi has been supported by grants from
Furuhashi M, Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.Nat Rev Drug Discov. 2008; 7:489–503. doi: 10.1038/nrd2589.CrossrefMedlineGoogle Scholar
Furuhashi M, Ishimura S, Ota H, Miura T. Lipid chaperones and metabolic inflammation.Int J Inflam. 2011; 2011:642612. doi: 10.4061/2011/642612.CrossrefMedlineGoogle Scholar
Furuhashi M, Saitoh S, Shimamoto K, Miura T. Fatty Acid-Binding Protein 4 (FABP4): Pathophysiological Insights and Potent Clinical Biomarker of Metabolic and Cardiovascular Diseases.Clin Med Insights Cardiol. 2014; 8(suppl 3):23–33. doi: 10.4137/CMC.S17067.MedlineGoogle Scholar
Hotamisligil GS, Johnson RS, Distel RJ, Ellis R, Papaioannou VE, Spiegelman BM. Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein.Science. 1996; 274:1377–1379.CrossrefMedlineGoogle Scholar
Makowski L, Boord JB, Maeda K, Babaev VR, Uysal KT, Morgan MA, Parker RA, Suttles J, Fazio S, Hotamisligil GS, Linton MF. Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis.Nat Med. 2001; 7:699–705. doi: 10.1038/89076.CrossrefMedlineGoogle Scholar
Furuhashi M, Fucho R, Görgün CZ, Tuncman G, Cao H, Hotamisligil GS. Adipocyte/macrophage fatty acid-binding proteins contribute to metabolic deterioration through actions in both macrophages and adipocytes in mice.J Clin Invest. 2008; 118:2640–2650. doi: 10.1172/JCI34750.MedlineGoogle Scholar
Boord JB, Maeda K, Makowski L, Babaev VR, Fazio S, Linton MF, Hotamisligil GS. Combined adipocyte-macrophage fatty acid-binding protein deficiency improves metabolism, atherosclerosis, and survival in apolipoprotein E-deficient mice.Circulation. 2004; 110:1492–1498. doi: 10.1161/01.CIR.0000141735.13202.B6.LinkGoogle Scholar
Furuhashi M, Tuncman G, Görgün CZ, Makowski L, Atsumi G, Vaillancourt E, Kono K, Babaev VR, Fazio S, Linton MF, Sulsky R, Robl JA, Parker RA, Hotamisligil GS. Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2.Nature. 2007; 447:959–965. doi: 10.1038/nature05844.CrossrefMedlineGoogle Scholar
Xu A, Wang Y, Xu JY, Stejskal D, Tam S, Zhang J, Wat NM, Wong WK, Lam KS. Adipocyte fatty acid-binding protein is a plasma biomarker closely associated with obesity and metabolic syndrome.Clin Chem. 2006; 52:405–413. doi: 10.1373/clinchem.2005.062463.CrossrefMedlineGoogle Scholar
Cao H, Sekiya M, Ertunc ME, Burak MF, Mayers JR, White A, Inouye K, Rickey LM, Ercal BC, Furuhashi M, Tuncman G, Hotamisligil GS. Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production.Cell Metab. 2013; 17:768–778. doi: 10.1016/j.cmet.2013.04.012.CrossrefMedlineGoogle Scholar
Mita T, Furuhashi M, Hiramitsu S,. FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms.Obesity (Silver Spring). 2015; 23:359–367. doi: 10.1002/oby.20954.CrossrefMedlineGoogle Scholar
Lamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U, Schunck WH, Ehrhart-Bornstein M, Bornstein SR, Morano I. Adipocyte fatty acid-binding protein suppresses cardiomyocyte contraction: a new link between obesity and heart disease.Circ Res. 2009; 105:326–334. doi: 10.1161/CIRCRESAHA.109.200501.LinkGoogle Scholar
Xu A, Tso AW, Cheung BM, Wang Y, Wat NM, Fong CH, Yeung DC, Janus ED, Sham PC, Lam KS. Circulating adipocyte-fatty acid binding protein levels predict the development of the metabolic syndrome: a 5-year prospective study.Circulation. 2007; 115:1537–1543. doi: 10.1161/CIRCULATIONAHA.106.647503.LinkGoogle Scholar
von Eynatten M, Breitling LP, Roos M, Baumann M, Rothenbacher D, Brenner H. Circulating adipocyte fatty acid-binding protein levels and cardiovascular morbidity and mortality in patients with coronary heart disease: a 10-year prospective study.Arterioscler Thromb Vasc Biol. 2012; 32:2327–2335. doi: 10.1161/ATVBAHA.112.248609.LinkGoogle Scholar
Ota H, Furuhashi M, Ishimura S, Koyama M, Okazaki Y, Mita T, Fuseya T, Yamashita T, Tanaka M, Yoshida H, Shimamoto K, Miura T. Elevation of fatty acid-binding protein 4 is predisposed by family history of hypertension and contributes to blood pressure elevation.Am J Hypertens. 2012; 25:1124–1130. doi: 10.1038/ajh.2012.88.CrossrefMedlineGoogle Scholar
Ishimura S, Furuhashi M, Watanabe Y, Hoshina K, Fuseya T, Mita T, Okazaki Y, Koyama M, Tanaka M, Akasaka H, Ohnishi H, Yoshida H, Saitoh S, Miura T. Circulating levels of fatty acid-binding protein family and metabolic phenotype in the general population.PLoS One. 2013; 8:e81318. doi: 10.1371/journal.pone.0081318.CrossrefMedlineGoogle Scholar
Furuhashi M, Ishimura S, Ota H, Hayashi M, Nishitani T, Tanaka M, Yoshida H, Shimamoto K, Hotamisligil GS, Miura T. Serum fatty acid-binding protein 4 is a predictor of cardiovascular events in end-stage renal disease.PLoS One. 2011; 6:e27356. doi: 10.1371/journal.pone.0027356.CrossrefMedlineGoogle Scholar
Fuseya T, Furuhashi M, Yuda S,. Elevation of circulating fatty acid-binding protein 4 is independently associated with left ventricular diastolic dysfunction in a general population.Cardiovasc Diabetol. 2014; 13:126. doi: 10.1186/s12933-014-0126-7.CrossrefMedlineGoogle Scholar
Doi M, Miyoshi T, Hirohata S, Nakamura K, Usui S, Takeda K, Iwamoto M, Kusachi S, Kusano K, Ito H. Association of increased plasma adipocyte fatty acid-binding protein with coronary artery disease in non-elderly men.Cardiovasc Diabetol. 2011; 10:44. doi: 10.1186/1475-2840-10-44.CrossrefMedlineGoogle Scholar
Agardh HE, Folkersen L, Ekstrand J, Marcus D, Swedenborg J, Hedin U, Gabrielsen A, Paulsson-Berne G. Expression of fatty acid-binding protein 4/aP2 is correlated with plaque instability in carotid atherosclerosis.J Intern Med. 2011; 269:200–210. doi: 10.1111/j.1365-2796.2010.02304.x.CrossrefMedlineGoogle Scholar
Peeters W, de Kleijn DP, Vink A, van de Weg S, Schoneveld AH, Sze SK, van der Spek PJ, de Vries JP, Moll FL, Pasterkamp G. Adipocyte fatty acid binding protein in atherosclerotic plaques is associated with local vulnerability and is predictive for the occurrence of adverse cardiovascular events.Eur Heart J. 2011; 32:1758–1768. doi: 10.1093/eurheartj/ehq387.CrossrefMedlineGoogle Scholar
Sacks HS, Fain JN. Human epicardial adipose tissue: a review.Am Heart J. 2007; 153:907–917. doi: 10.1016/j.ahj.2007.03.019.CrossrefMedlineGoogle Scholar
Itakura H, Yokoyama M, Matsuzaki M,.; JELIS Investigators. Relationships between plasma fatty acid composition and coronary artery disease.J Atheroscler Thromb. 2011; 18:99–107.CrossrefMedlineGoogle Scholar
Ho E, Shimada Y. Formation of the epicardium studied with the scanning electron microscope.Dev Biol. 1978; 66:579–585.CrossrefMedlineGoogle Scholar
Aghamohammadzadeh R, Withers S, Lynch F, Greenstein A, Malik R, Heagerty A. Perivascular adipose tissue from human systemic and coronary vessels: the emergence of a new pharmacotherapeutic target.Br J Pharmacol. 2012; 165:670–682. doi: 10.1111/j.1476-5381.2011.01479.x.CrossrefMedlineGoogle Scholar
Ishii T, Asuwa N, Masuda S, Ishikawa Y. The effects of a myocardial bridge on coronary atherosclerosis and ischaemia.J Pathol. 1998; 185:4–9.CrossrefMedlineGoogle Scholar
Wisneski JA, Gertz EW, Neese RA, Mayr M. Myocardial metabolism of free fatty acids. Studies with 14C-labeled substrates in humans.J Clin Invest. 1987; 79:359–366. doi: 10.1172/JCI112820.CrossrefMedlineGoogle Scholar
Nelson RH, Prasad A, Lerman A, Miles JM. Myocardial uptake of circulating triglycerides in nondiabetic patients with heart disease.Diabetes. 2007; 56:527–530. doi: 10.2337/db06-1552.CrossrefMedlineGoogle Scholar
Marchington JM, Mattacks CA, Pond CM. Adipose tissue in the mammalian heart and pericardium: structure, foetal development and biochemical properties.Comp Biochem Physiol B. 1989; 94:225–232.CrossrefMedlineGoogle Scholar
Marchington JM, Pond CM. Site-specific properties of pericardial and epicardial adipose tissue: the effects of insulin and high-fat feeding on lipogenesis and the incorporation of fatty acids in vitro.Int J Obes. 1990; 14:1013–1022.MedlineGoogle Scholar
Vural B, Atalar F, Ciftci C, Demirkan A, Susleyici-Duman B, Gunay D, Akpinar B, Sagbas E, Ozbek U, Buyukdevrim AS. Presence of fatty-acid-binding protein 4 expression in human epicardial adipose tissue in metabolic syndrome.Cardiovasc Pathol. 2008; 17:392–398. doi: 10.1016/j.carpath.2008.02.006.CrossrefMedlineGoogle Scholar
Masoodi M, Kuda O, Rossmeisl M, Flachs P, Kopecky J. Lipid signaling in adipose tissue: Connecting inflammation & metabolism.Biochim Biophys Acta. 2015; 1851:503–518. doi: 10.1016/j.bbalip.2014.09.023.CrossrefMedlineGoogle Scholar
Makowski L, Brittingham KC, Reynolds JM, Suttles J, Hotamisligil GS. The fatty acid-binding protein, aP2, coordinates macrophage cholesterol trafficking and inflammatory activity. Macrophage expression of aP2 impacts peroxisome proliferator-activated receptor gamma and IkappaB kinase activities.J Biol Chem. 2005; 280:12888–12895. doi: 10.1074/jbc.M413788200.CrossrefMedlineGoogle Scholar
Fu Y, Luo N, Lopes-Virella MF. Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages.J Lipid Res. 2000; 41:2017–2023.CrossrefMedlineGoogle Scholar
Shum BO, Mackay CR, Gorgun CZ, Frost MJ, Kumar RK, Hotamisligil GS, Rolph MS. The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation.J Clin Invest. 2006; 116:2183–2192. doi: 10.1172/JCI24767.CrossrefMedlineGoogle Scholar
Grimsrud PA, Picklo MJ, Griffin TJ, Bernlohr DA. Carbonylation of adipose proteins in obesity and insulin resistance: identification of adipocyte fatty acid-binding protein as a cellular target of 4-hydroxynonenal.Mol Cell Proteomics. 2007; 6:624–637. doi: 10.1074/mcp.M600120-MCP200.CrossrefMedlineGoogle Scholar
Hitomi M, Odani S, Ono T. Glutathione-protein mixed disulfide decreases the affinity of rat liver fatty acid-binding protein for unsaturated fatty acid.Eur J Biochem. 1990; 187:713–719.CrossrefMedlineGoogle Scholar
Smathers RL, Fritz KS, Galligan JJ, Shearn CT, Reigan P, Marks MJ, Petersen DR. Characterization of 4-HNE modified L-FABP reveals alterations in structural and functional dynamics.PLoS One. 2012; 7:e38459. doi: 10.1371/journal.pone.0038459.CrossrefMedlineGoogle Scholar
Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stress in obesity and its impact on metabolic syndrome.J Clin Invest. 2004; 114:1752–1761. doi: 10.1172/JCI21625.CrossrefMedlineGoogle Scholar
Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance.J Clin Invest. 2006; 116:3015–3025. doi: 10.1172/JCI28898.CrossrefMedlineGoogle Scholar
Girona J, Rosales R, Plana N, Saavedra P, Masana L, Vallvé JC. FABP4 induces vascular smooth muscle cell proliferation and migration through a MAPK-dependent pathway.PLoS One. 2013; 8:e81914. doi: 10.1371/journal.pone.0081914.CrossrefMedlineGoogle Scholar
Aragonès G, Saavedra P, Heras M, Cabré A, Girona J, Masana L. Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells.Cardiovasc Diabetol. 2012; 11:72. doi: 10.1186/1475-2840-11-72.CrossrefMedlineGoogle Scholar
Wu LE, Samocha-Bonet D, Whitworth PT, Fazakerley DJ, Turner N, Biden TJ, James DE, Cantley J. Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity.Mol Metab. 2014; 3:465–473. doi: 10.1016/j.molmet.2014.02.005.CrossrefMedlineGoogle Scholar
Chow WS, Tso AW, Xu A, Yuen MM, Fong CH, Lam TH, Lo SV, Tse HF, Woo YC, Yeung CY, Cheung BM, Lam KS. Elevated circulating adipocyte-fatty acid binding protein levels predict incident cardiovascular events in a community-based cohort: a 12-year prospective study.J Am Heart Assoc. 2013; 2:e004176. doi: 10.1161/JAHA.112.004176.LinkGoogle Scholar
Furuhashi M, Mita T, Moniwa N, Hoshina K, Ishimura S, Fuseya T, Watanabe Y, Yoshida H, Shimamoto K, Miura T. Angiotensin II receptor blockers decrease serum concentration of fatty acid-binding protein 4 in patients with hypertension.Hypertens Res. 2015; 38:252–259. doi: 10.1038/hr.2015.2.CrossrefMedlineGoogle Scholar
Karpisek M, Stejskal D, Kotolova H, Kollar P, Janoutova G, Ochmanova R, Cizek L, Horakova D, Yahia RB, Lichnovska R, Janout V. Treatment with atorvastatin reduces serum adipocyte-fatty acid binding protein value in patients with hyperlipidaemia.Eur J Clin Invest. 2007; 37:637–642. doi: 10.1111/j.1365-2362.2007.01835.x.CrossrefMedlineGoogle Scholar
Furuhashi M, Hiramitsu S, Mita T, Fuseya T, Ishimura S, Omori A, Matsumoto M, Watanabe Y, Hoshina K, Tanaka M, Moniwa N, Yoshida H, Ishii J, Miura T. Reduction of serum FABP4 level by sitagliptin, a DPP-4 inhibitor, in patients with type 2 diabetes mellitus.J Lipid Res. 2015; 56:2372–2380. doi: 10.1194/jlr.M059469.CrossrefMedlineGoogle Scholar
Furuhashi M, Hiramitsu S, Mita T, Omori A, Fuseya T, Ishimura S, Watanabe Y, Hoshina K, Matsumoto M, Tanaka M, Moniwa N, Yoshida H, Ishii J, Miura T. Reduction of circulating FABP4 level by treatment with omega-3 fatty acid ethyl esters.Lipids Health Dis. 2016; 15:5. doi: 10.1186/s12944-016-0177-8.CrossrefMedlineGoogle Scholar
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.