Ectopic Fatty Acid–Binding Protein 4 Expression in the Vascular Endothelium is Involved in Neointima Formation After Vascular Injury
Background Fatty acid‐binding protein 4 (FABP4) is expressed in adipocytes, macrophages, and endothelial cells of capillaries but not arteries. FABP4 is secreted from adipocytes in association with lipolysis, and an elevated circulating FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the link between FABP4 and endovascular injury. We investigated the involvement of ectopic FABP4 expression in endothelial cells in neointima hyperplasia after vascular injury.
Methods and Results Femoral arteries of 8‐week‐old male mice were subjected to wire‐induced vascular injury. After 4 weeks, immunofluorescence staining showed that FABP4 was ectopically expressed in endothelial cells of the hyperplastic neointima. Neointima formation determined by intima area and intima to media ratio was significantly decreased in FABP4‐defficient mice compared with that in wild‐type mice. Adenovirus‐mediated overexpression of FABP4 in human coronary artery endothelial cells (HCAECs) in vitro increased inflammatory cytokines and decreased phosphorylation of nitric oxide synthase 3. Furthermore, FABP4 was secreted from HCAECs. Treatment of human coronary smooth muscle cells or HCAECs with the conditioned medium of Fabp4‐overexpressed HCAECs or recombinant FABP4 significantly increased gene expression of inflammatory cytokines and proliferation‐ and adhesion‐related molecules in cells, promoted cell proliferation and migration of human coronary smooth muscle cells, and decreased phosphorylation of nitric oxide synthase 3 in HCAECs, which were attenuated in the presence of an anti‐FABP4 antibody.
Conclusions Ectopic expression and secretion of FABP4 in vascular endothelial cells contribute to neointima formation after vascular injury. Suppression of ectopic FABP4 in the vascular endothelium would be a novel strategy against post‐angioplasty vascular restenosis.
- endothelial cell
- fatty acid–binding protein
- neointimal hyperplasia
- smooth muscle cell
- vascular inflammation
- vascular remodeling
What Is New?
Ectopic expression of fatty acid‐binding protein 4 (FABP4) in vascular endothelial cells and accompanying inflammation were associated with neointima formation in the artery after wire‐induced endovascular injury.
FABP4 was secreted from vascular endothelial cells.
Secreted FABP4 derived from ectopic FABP4 expression in endothelial cells promoted proliferative and migratory responses of vascular smooth muscle cells and vascular endothelial dysfunction, which were cancelled by the use of anti‐FABP4 antibody.
What Are the Clinical Implications?
Ectopic expression and secretion of FABP4 in the vascular endothelium are associated with neointima formation after vascular injury through endothelial dysfunction and inflammation in vascular endothelial cells and proliferation, migration, and inflammation in vascular smooth muscle cells.
Inhibition of FABP4 by a small molecule, neutralization of FABP4 by the use of an antibody, or blocking unidentified receptors of FABP4 would be beneficial for the prevention of post‐angioplasty vascular restenosis.
Neointima hyperplasia in coronary artery lesions subjected to percutaneous coronary intervention is a pathologic characteristic of restenosis.1 Endovascular injury causes impairment and regeneration of endothelial cells and activates vascular smooth muscle cells and smooth muscle cell–like cells differentiated from bone marrow–derived progenitor cells in the media, leading to neointima formation by cell migration into the intima and proliferation.2, 3 Drug‐eluting stents have proven to be more effective than bare metal stents in reducing the incidence of restenosis after percutaneous coronary intervention, but coronary restenosis and stent thrombosis remain significant problems.4
Fatty acid–binding proteins are 14‐ to 15‐kDa cytosolic proteins that can reversibly bind to saturated and unsaturated long‐chain fatty acids with high affinity.5, 6, 7 It has been proposed that fatty acid–binding proteins facilitate the transport of lipids to specific compartments in the cell. Fatty acid‐binding protein 4 (FABP4), known as adipocyte fatty acid–binding protein, or aP2, is expressed in both adipocytes and macrophages and plays an important role in the development of insulin resistance and atherosclerosis.8, 9, 10 We previously demonstrated that inhibition of FABP4 in cells would be a novel therapeutic strategy against insulin resistance, diabetes mellitus, and atherosclerosis.11
It has recently been reported that FABP4 is secreted from adipocytes in association with lipolysis via a nonclassical secretion pathway,12, 13 although there are no typical secretory signal peptides in the sequence of FABP4.5 FABP4 has also been shown to be secreted from macrophages,14 although the predominant contributors of circulating FABP4 are adipocytes rather than macrophages.12 Previous studies demonstrated that circulating FABP4 acts as an adipokine, an adipocyte‐derived bioactive molecule, for the development of insulin resistance and atherosclerosis.12, 14 Furthermore, elevation of the circulating FABP4 level is associated with obesity, insulin resistance, hypertension, cardiac dysfunction, dyslipidemia, and atherosclerosis.15, 16, 17, 18, 19, 20, 21 Several drugs for dyslipidemia, diabetes mellitus, and hypertension have been reported to modulate FABP4 levels.22, 23, 24, 25, 26, 27, 28
Other than adipocytes and macrophages, FABP4 is also expressed in endothelial cells of capillaries and small veins, but not arteries, in several mouse and human tissues including the heart and kidney.29, 30 We previously demonstrated that ectopic FABP4 expression in endothelial cells of the glomerulus is associated with progression of proteinuria and renal dysfunction31 and that urinary excretion of FABP4 would be a novel biomarker of glomerular damage.32 Interestingly, it has been reported that FABP4 is markedly and ectopically upregulated in artery endothelial cells regenerating after endothelial balloon denudation in the pig coronary artery.33 However, little is known about the link between FABP4 and endovascular injury. We therefore investigated the involvement of ectopic FABP4 expression in endothelial cells of neointima hyperplasia after endovascular injury by using a mouse model of wire‐induced femoral artery injury, which mimics vascular remodeling following coronary angioplasty,34 and by performing in vitro experiments using co‐culture models and treatment with recombinant FABP4 and anti‐FABP4 antibody in vascular endothelial cells and smooth muscle cells.
Biochemical Reagents and Animals
All experimental protocols were approved by the Animal Care Committee of Sapporo Medical University, and animal care and experimental procedures were performed in accordance with the Animal Care Committee of Sapporo Medical University. All biochemical reagents were purchased from Sigma‐Aldrich unless indicated otherwise. Male C57BL/6J mice were obtained from Oriental Yeast. FABP4‐deficient (Fabp4−/−) and wild‐type (Fabp4+/+) mice were generated in the laboratory of Dr Gökhan S. Hotamisligil (Harvard T.H. Chan School of Public Health) and backcrossed for more than 8 generations into the C56BL/6J genetic background. Mice were kept on a 12‐hour light cycle in a pathogen‐free barrier facility and were placed on a regular chow diet ad libitum.
Wire Injury Model
Male mice aged 8 weeks were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg), and endoluminal injury of the left femoral artery was performed as previously described.34, 35 In brief, the left femoral artery was exposed and clamped upstream and downstream of the artery. From the branch of the femoral artery, a wire (diameter: 0.014 inches) was inserted into the artery and removed from the vessel followed by branch ligation.
Histological analysis was performed as previously described.35, 36 At 4 weeks after wire‐induced injury, mice were anesthetized and fixed by perfusion with saline followed by 10% formalin through a cannula placed in the left ventricle. Both femoral arteries were excised from each mouse and embedded in paraffin. Neointima formation in the femoral arteries was evaluated at 10 locations at 100‐μm intervals, with the most distal site located at the origin of the branch through which the wire had been inserted. The sections were stained by the Elastica‐Van Gieson protocol. Images of arteries were captured with a digital color camera (DP21, Olympus) mounted on a microscope (CX41, Olympus). Quantitative analysis for areas of the intima and media was performed using ImageJ software. All of the measurements were performed in a double‐blind manner by 2 different researchers.
Immunofluorescence staining using rabbit anti‐FABP4 (Abcam) and mouse anit‐CD31 (Abcam) antibodies was performed as previously described.35, 36 Control experiments were performed by omitting the primary antibodies. Images were captured with a fluorescent microscope (BIOREVO BZ‐9000 with a BZ‐II analyzer, Keyence) or an LSM510META ConfoCor3 microscope (Carl Zeiss).
Quantitative Real‐Time PCR
Total RNA was isolated using Trizol Reagent (Invitrogen). One microgram of total RNA was reverse‐transcribed by using the High‐Capacity cDNA Archive Kit (Applied Biosystems). Quantitative real‐time PCR analysis was performed using SYBR Green in the real‐time PCR system (Applied Biosystems). The thermal cycling program was 10 minutes at 95°C for enzyme activation and 40 cycles of denaturation for 15 seconds at 95°C, 30‐second annealing at 58°C, and 30‐second extension at 72°C. Primers used in the present study are listed in Table. To normalize expression data, 18s rRNA was used as an internal control gene.
Production and Purification of Recombinant FABP4 and Anti‐FABP4 Antibody
Recombinant mouse FABP4 with a 6× His tag was produced in Escherichia coli using the pET21a vector (Novagen) and was purified with HisTrap HP (GE Healthcare) followed by endotoxin removal with a commercial system (Millipore). The rabbit polyclonal antibody against mouse FABP4 was produced using the recombinant full‐length FABP4 protein, and the antibody was purified from the serum of the final bleed using the NAb Spin system (Pierce Biotechnology, Inc). Preimmune serum was purified similarly and used as a control.
Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Lonza. HCAECs and HCASMCs were grown in Endothelial Cell Basal Medium‐2 (EBM‐2; Lonza) supplemented with EGM‐2 MV SingleQuots (Lonza) and in Smooth Muscle Cell Basal Medium (Lonza) supplemented with SmGM‐2 SingleQuots (Lonza), respectively, according to the manufacturer's instructions. After serum starvation for 24 hours, cells were stimulated with 50 ng/mL of vascular endothelial growth factor (VEGF) (R&D Systems) for 0.5 or 24 hours, 0.5 μmol/L insulin for 0.5 hours, 100 μmol/L H2O2 for 1 hour followed by 23‐hour incubation in normal culture medium after washing, 0 to 200 nmol/L recombinant FABP4, or 10 μg/mL anti‐FABP4 antibody in the medium supplemented with 0.5% BSA. The doses of reagents and incubation periods varied according to the experimental protocol. Each experiment was performed in at least triplicate.
Overexpression of FABP4 in HCAECs
HCAECs were infected with an adenovirus green fluorescent protein expressing vector‐transfected human cDNA of FABP4 (Ad‐FABP4) or an empty sequence (Ad‐Control) as a control, which had been generated by Sirion Biotech. The cells were infected with the adenoviruses at a multiplicity of infection of 100 and a transduction enhancer (AdenoBoost, Sirion Biotech), and experiments were performed 2 days after infection. The conditioned medium (CM) was prepared by 24‐hour incubation of HACECs transfected with Ad‐FABP4 or Ad‐Control in EBM‐2 supplemented with 0.1% or 5% FBS or 0.5% BSA. Co‐culture experiments were performed for HCASMCs and HCAECs treated with the CM of Ad‐FABP4– and Ad‐Control–transfected HCAECs or coincubated with Ad‐FABP4– and Ad‐Control–transfected HCAECs using insert transparent wells of a 1.0‐μm pore size membrane (Falcon).
Assessment of FABP4 Secretion From Cultured HCAECs
After overnight serum depletion by 0.5% BSA in the medium, HCAECs were incubated with or without 10 μmol/L isopropanol in the medium supplemented with 0.5% BSA for 2 to 24 hours. The CM from the cells was filtered to obtain a 10‐ to 50‐kDa fraction of proteins using Amicon Ultra 10K and 50K devices (Millipore). Total protein content of the cell lysate (CL) in a cell lysis buffer, containing 50 mmol/L Tris‐HCl (pH 7.0), 2 mmol/L EGTA, 5 mmol/L EDTA, 30 mmol/L NaF, 10 mmol/L Na3VO4, 10 mmol/L Na4P2O7, 40 mmol/L β‐glycerophosphate, 0.5% NP‐40, and 1% protease inhibitor cocktail, was assessed by a microplate protein assay based on Lowry's method (Bio‐Rad). FABP4 protein in the CL and CM was determined by Western blotting and analyzed as previously described.13, 28
FABP4 level in the CM was also measured using a commercially available enzyme‐linked immunosorbent assay kit for FABP4 (Biovendor R&D). The intra‐assay and interassay coefficients of variation in the kits were <5%. According to the manufacturer's protocol, no cross‐reactivity of FABP4 with other fatty acid–binding protein types was observed. Secretion of FABP4 into the CM was normalized to total protein concentration of the CL.
Total protein content of the samples was assessed by a microplate protein assay based on Lowry's method, and equal amounts of protein per sample and known molecular weight markers were subjected to SDS‐PAGE. Proteins were electrophoretically transferred onto polyvinylidene fluoride membranes and incubated for 1 hour at room temperature with a blocking solution (3% BSA) in Tris‐buffered saline buffer containing 0.1% Tween 20. The blocked membranes were incubated with primary antibodies for FABP4 (Abcam), GAPDH (Santa Cruz Biotechnology), nitric oxide synthase 3 (NOS3; BD Biosciences), phosphorylated NOS3 (BD Biosciences), and actin overnight at 4°C and washed 3 times with Tris‐buffered saline buffer with 0.1% Tween 20. The membranes were incubated with a secondary antibody conjugated with horseradish peroxidase (GE Healthcare) for 1 hour at room temperature and washed. Immunodetection analyses were performed using a BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Diagnostics). Densitometry was analyzed using ImageJ software.
Cell Proliferation and Migration Assays
An MTS assay was performed for assessing cell proliferation using the cell titer Aqueous One Solution Proliferation Assay (Promega). After overnight serum starvation by Smooth Muscle Cell Basal Medium with 0.1% FBS, HCASMCs were stimulated with the CM prepared by 24‐hour incubation of Ad‐FABP4– and Ad‐Control–transfected HCAECs in EBM‐2 supplemented with 5% FBS for 24 hours.
Cell proliferation was also analyzed by measuring DNA synthesis with a colorimetric bromodeoxyuridine (BrdU) enzyme‐linked immunosorbent assay kit (Roche Diagnostics) according to the manufacturer's instructions. Briefly, 1×104 HCASMCs were seeded in a 96‐well microplate and cultured with the CM prepared by 24‐hour incubation of Ad‐FABP4– and Ad‐Control–transfected HCAECs in EBM‐2 supplemented with 0.1% FBS or 0 to 200 nmol/L recombinant FABP4 supplemented with 0.1% FBS in the presence of 0 to 10 μg/mL anti‐FABP4 antibody for 24 hours. The cells were then labeled with BrdU labeling reagent for 10 hours. After fixation, the cells were incubated with anti‐BrdU antibody for 1.5 hours. After washing, 100 μL of a substrate (tetramethylbenzidine) was added to each well and the plates were incubated at room temperature for 0.5 hours. The absorbance at 450 nm was measured with an ELISA reader (Synergy H4, Biotek).
A scratch wound assay was performed for determining cell migration. After overnight serum starvation by Smooth Muscle Cell Basal Medium with 0.5% BSA, HCASMCs were scratched with a sterile pipette tip to produce a straight cell‐free zone. The cells were stimulated with the CM prepared by 24‐hour incubation of Ad‐FABP4– and Ad‐Control–transfected HCAECs in EBM‐2 supplemented with 5% FBS or 100 nmol/L recombinant FABP4 supplemented with 0.5% BSA in the presence and absence of 10 μg/mL anti‐FABP4 antibody for 15 hours. Pictures were taken at baseline and after stimulation, and migration distance was measured using ImageJ software as previously described.35
Numeric variables are expressed as means±SEM. After confirming normal distribution of each variable, differences of means were analyzed using 1‐way ANOVA with the Tukey‐Kramer post hoc test for multiple groups and the Student t test for 2 groups. P values <0.05 were considered significant. All data were analyzed using JMP 9 for Macintosh (SAS Institute Inc).
Involvement of Ectopic FABP4 Expression in Endothelial Cells in Neointima Formation
The left femoral arteries of 8‐week‐old male Fabp4+/+ and Fabp4−/− mice were subjected to wire‐induced vascular injury to investigate the association between vascular remodeling and ectopic expression of FABP4 in vivo. Four weeks later, wire injury‐mediated neointima hyperplasia was induced, and the thickness of the neointima determined by intima area and intima to media ratio was significantly smaller in FABP4−/− mice than in Fabp4+/+ mice (Figure 1A through 1C). Gene expression levels of fatty acid–binding protein 5 were comparable in the injured arteries of Fabp4+/+ and Fabp4−/− mice, but Fabp4−/− mice had significantly lower gene expression levels of inflammatory cytokines in the injured arteries, including MCP‐1 (monocyte chemotactic protein‐1), interleukin 1β (IL‐1β), interleukin 6 (IL‐6), and tumor necrosis factor α (TNF‐α), than did Fabp4+/+ mice (Figure 1D). Immunofluorescence staining showed that FABP4 was absent in noninjured arteries of Fabp4+/+ and Fabp4−/− mice but was present in cells stained by CD31, a maker of endothelial cells, of the hyperplastic neointima in the injured arteries of Fabp4+/+ mice (Figure 1E).
Effects of FABP4 Overexpression in Vascular Endothelial Cells
Gene (Figure 2A) and protein (Figure 2B) expressions of FABP4 were induced by treatment of HCAECs with VEGF and H2O2 as previously reported in endothelial cells.29, 37 Adenovirus‐mediated Fabp4 overexpression in HCAECs (Figure 2C) resulted in efficient gene and protein induction of FABP4 (Figure 2D and 2E) and decreased phosphorylation of NOS3 by stimulation with insulin or VEGF (Figure 2F). Overexpression of Fabp4 in HCAECs significantly increased gene expression levels of inflammatory cytokines, including Mcp1, Il1b, Il6, and Tnfa (Figure 2G), and adhesion‐related molecules, including intracellular adhesion molecule–1 (ICAM‐1), selectin E (SELE), integrin α 5 (ITGA5), and integrin β 3 (ITGB3) (Figure 2H).
Secretion of FABP4 From Vascular Endothelial Cells
Results from Western blot analysis showed that FABP4 was present in both the CL and CM of HCAECs and that GAPDH, a nonsecretory protein, was not present in the CM (Figure 2I), indicating that FABP4 in the CM was a result of its secretion from HCAECs, not a result of its leakage via injured cell membranes. FABP4 was secreted from HCAECs in a time‐dependent manner (Figure 2I), and the secretion of FABP4 was slightly, but significantly, enhanced in the presence of isoproterenol for 12 and 24 hours, which is known to increase secretion of FABP4 from adipocytes.12, 13 Secretion of FABP4 in the CM of Ad‐FABP4–transfected HCAECs was significantly greater than that in the CM of Ad‐Control–transfected HCAECs (Figure 2J).
Effects of HCASMCs Coincubated With Fabp4‐Overexpressed HCAEC
Incubation of HCASMCs with the CM of Fabp4‐overexpressed HCAECs (Figure 3A) significantly increased gene expression levels of inflammatory cytokines, including Mcp1, Il1b, Il6, and Tnfa; proliferation‐related molecules, including platelet‐derived growth factor receptor α and β; and adhesion‐related molecules, including Itga5 and Itgb3, which were attenuated in the presence of anti‐FABP4 antibody (Figure 3B). MTS and BrdU assays showed that incubation of HCASMCs with the CM of Fabp4‐overexpressed HCAECs significantly increased cell proliferation, which was cancelled by coincubation with anti‐FABP4 antibody (Figure 3C and 3D). The scratch wound–healing assay showed that HCASMCs incubated with the CM of Fabp4‐overexpressed HCAECs migrated faster than did those incubated with the CM of control cells, which was cancelled by coincubation with anti‐FABP4 antibody (Figure 3E).
In HCASMCs coincubated with Fabp4‐overexpressed HCAECs using insert transparent wells (Figure 3F), similar results were obtained for gene expression levels of inflammatory cytokines (Figure 3G) and proliferation‐ and adhesion‐related molecules (Figure 3H).
Treatment of HCASMCs With Recombinant FABP4 and Anti‐FABP4 Antibody
Treatment of HCASMCs with recombinant FABP4 (Figure 4A; HCASMC‐Rec) significantly increased gene expression levels of inflammatory cytokines and proliferation‐ and adhesion‐related molecules (Figure 4B), which were attenuated in the presence of anti‐FABP4 antibody (Figure 4C). Western blot analysis showed that recombinant FABP4 with a 6× His tag was partially internalized into HCASMCs, although endogenous expression of FABP4 was absent in HCASMCs (Figure 4D), indicating possible effects of exogenous FABP4 on HACSMCs in both extracellular and intracellular manners. A BrdU assay showed that treatment of HCASMCs with recombinant FABP4 significantly increased cell proliferation, which was attenuated by coincubation with anti‐FABP4 antibody in a dose‐dependent manner (Figure 4E). The scratch wound–healing assay showed that FABP4‐treated HCASMCs migrated faster than did untreated cells, which was cancelled by coincubation with anti‐FABP4 antibody (Figure 4F).
Effects of HCAECs Coincubated With Fabp4‐Overexpressed HCAECs
Incubation of HCAECs with the CM of Fabp4‐overexpressed HCAECs (Figure 5A) significantly increased gene expression levels of inflammatory cytokines, including Mcp1, Il1b, Il6, and Tnfa, and adhesion‐related molecules, including Icam1, Sele Itga5, and Itgb3 (Figure 5B), which were attenuated in the presence of anti‐FABP4 antibody (Figure 5C).
Similarly, in HCAECs coincubated with Fabp4‐overexpressed HCAECs using insert transparent wells (Figure 5D), gene expression levels of inflammatory cytokines (Figure 5E) and adhesion‐related molecules (Figure 5F) were increased.
Treatment of HCAECs With Recombinant FABP4 and Anti‐FABP4 Antibody
Treatment of HCAECs with recombinant FABP4 (Figure 6A) increased gene expression levels of inflammatory cytokines, including Mcp1, Il1b, Il6, and Tnfa, and adhesion‐related molecules, including Icam1, Sele, Itga5, and Itgb3 (Figure 6B), which were attenuated by coincubation with anti‐FABP4 antibody (Figure 6C). Similarly in HCASMCs, findings from Western blot analysis showed that recombinant FABP4 with a 6× His tag was partially internalized into HCAECs (Figure 6D), indicating possible effects of exogenous FABP4 on HCAECs in both extracellular and intracellular manners. Treatment of HCAECs with recombinant FABP4 decreased basal and VEGF‐stimulated phosphorylation of NOS3, and coincubation of recombinant FABP4 with anti‐FABP4 antibody restored VEGF‐stimulated phosphorylation of NOS3 (Figure 6E).
We demonstrated for the first time that ectopic expression of FABP4 in vascular endothelial cells and accompanying inflammation are associated with neointima formation in the artery after wire‐induced endovascular injury. FABP4 was ectopically induced in endothelial cells of the artery after endovascular injury as previously reported,33 and deletion of FABP4 significantly decreased neointima hyperplasia after wire‐induced vascular injury in vivo. In contrast, overexpression of Fabp4 in vascular endothelial cells increased the expression of inflammatory and adhesion‐related genes and impaired endothelial function. Furthermore, FABP4 was secreted from endothelial cells. Proliferative and migratory responses of HCASMCs and endothelial dysfunction of HCAECs were enhanced by treatment with the CM of Fabp4‐overexpressed endothelial cells or exogenous FABP4, which were cancelled by the use of anti‐FABP4 antibody. Thus, the present findings collectively suggest that secreted FABP4 derived from ectopic expression of FABP4 in endothelial cells after vascular injury contributes to the progression of neoinitima formation in paracrine and autocrine manners, resulting in vascular restenosis. The putative mechanism of secreted FABP4 derived from ectopic FABP4 expression in endothelial cells underlying the development of neointima formation is shown in Figure 7.
The pathology of neointima formation is traditionally thought to be migration and proliferation in the media, which are affected by various elements derived from around immune and vascular cells, such as growth factors and inflammatory cytokines.2 It has also been shown that smooth muscle cell–like cells differentiated from bone marrow–derived progenitor cells play important roles in neointima formation.3 Transcriptome and metabolome analyses showed that exogenous FABP4 affects transcriptional and metabolic regulation in adipose‐derived stem cells near adipocytes.38 Furthermore, previous studies using in vitro and in vivo experiments showed that circulating FABP4 acts as an adipokine, an adipocyte‐derived bioactive molecule, for the development of insulin resistance through increased hepatic glucose production12 and for the development of atherosclerosis through inhibition of endothelial NOS activity in endothelial cells,14, 39 proliferation and migration of vascular smooth muscle cells14, 40 and induction of inflammatory responses in macrophages, vascular smooth muscle cells, and vascular endothelial cells.14 Similarly, we demonstrated in the present study that treatment of vascular smooth muscle cells and vascular endothelial cells with recombinant FABP4 induced vascular inflammation, proliferative and migratory responses, and endothelial dysfunction. Recent studies have also demonstrated that neutralization of secreted FABP4 with an antibody to FABP4 could be a feasible approach for treatment of insulin resistance and type 2 diabetes mellitus.12, 41 In the present study, we revealed inhibitory effects of FABP4 neutralization using an antibody to FABP4 on proliferation, migration, and inflammatory response in vascular smooth muscle cells and on inflammatory response and endothelial dysfunction in vascular endothelial cells in vitro. Circulating FABP4 derived from ectopic expression of FABP4 in the endothelium may directly affect neointima formation after endovascular injury.
FABP4 is secreted from adipocytes under regulation by the catecholamine‐induced lipolytic signal pathway, although FABP4 lacks an N‐terminal secretory signal sequence.7, 12, 13 The present study showed that secretion of FABP4 from endothelial cells was slightly, but significantly, enhanced by stimulation of isoproterenol, similar to that in the case of adipocytes. It was previously shown that treatment with several inflammatory stimuli, including VEGF, H2O2 and cytokines, and cellular senescence induce expression of FABP4 in endothelial cells,29, 37 although the expression level of FABP4 in endothelial cells is lower than that in adipocytes.7, 30 It is possible that the level of FABP4 secreted from endothelial cells is sufficient for causing significant effects on nearby vascular endothelial cells and smooth muscle cells, leading to an inflammatory response, proliferation, and migration from the media into the intima. The circulating level of FABP4 is ≈20 ng/mL (1 nmol/L) in humans.19 Since autocrine and paracrine actions of the secreted FABP4 derived from regenerated endothelial cells were focused on in the present study, the concentration of recombinant FABP4 used (≈200 nmol/L) seems to be reasonable and physiological in the local area.
Evidence indicating that FABP4 acts as a biological molecule is accumulating,12, 14, 39, 40, 41, 42, 43 and serum FABP4 level has been reported to predict long‐term cardiovascular events.44, 45, 46 However, the receptor for FABP4 remains unknown. The present study showed that extracellular FABP4 is partially internalized into the cell, but it is unclear whether extracellular FABP4 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 as well as endovascular injury, such as neutralization of FABP4 and/or blockade of the FABP4 receptor, if any.
Ectopic expression and secretion of FABP4 in the vascular endothelium are associated with neointima formation after vascular injury through endothelial dysfunction and inflammation in vascular endothelial cells and proliferation, migration, and inflammation in vascular smooth muscle cells. A further understanding of ectopic expression of FABP4 from endothelial cells as well as adipocytes and macrophages may enable the development of new therapeutic strategies for cardiovascular and metabolic diseases. Inhibition of FABP4 by a small molecule, neutralization of FABP4 by the use of an antibody, or blocking unidentified receptors of FABP4 would be beneficial for the prevention of post‐angioplasty vascular restenosis.
Sources of Funding
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, Kondou Kinen Medical Foundation, and Terumo Foundation for Life Science and Arts.
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- ↵Mita T, Furuhashi M, Hiramitsu S, Ishii J, Hoshina K, Ishimura S, Fuseya T, Watanabe Y, Tanaka M, Ohno K, Akasaka H, Ohnishi H, Yoshida H, Saitoh S, Shimamoto K, Miura T. FABP4 is secreted from adipocytes by adenyl cyclase‐PKA‐ and guanylyl cyclase‐PKG‐dependent lipolytic mechanisms. Obesity (Silver Spring). 2015;23:359–367.
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- ↵Furuhashi M, Omori A, Matsumoto M, Kataoka Y, Tanaka M, Moniwa N, Ohnishi H, Yoshida H, Saitoh S, Shimamoto K, Miura T. Independent link between levels of proprotein convertase subtilisin/kexin type 9 and FABP4 in a general population without medication. Am J Cardiol. 2016;118:198–203.
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