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. 2009 Nov;23(11):3865–3873. doi: 10.1096/fj.09-134882

Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells

Harun Elmasri *, Cagatay Karaaslan *, Yaroslav Teper *, Elisa Ghelfi *, MeiQian Weng *, Tan A Ince †, Harry Kozakewich ‡, Joyce Bischoff §, Sule Cataltepe *,1
PMCID: PMC2775007  PMID: 19625659

Abstract

Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs.—Elmasri, H., Karaaslan, C., Teper, Y., Ghelfi, E., Weng, M., Ince, T. A., Kozakewich, H., Bischoff, J., Cataltepe, S. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.

Keywords: angiogenesis, infantile hemangioma, microvessel, placenta

Fatty acid binding protein 4 (FABP4; adipocyte-FABP; aP2) is a member of the family of intracellular FABPs (1). The FABP family consists of 9 highly conserved cytosolic proteins that are abundantly expressed in a tissue-specific manner with some overlap (2). FABPs are capable of binding a variety of hydrophobic ligands, such as long-chain fatty acids, eicosanoids, leukotrienes, and prostaglandins (3,4,5). Although the exact biological function of FABPs is not known, they have been implicated in several critical cellular processes, including uptake and trafficking of intracellular fatty acids and regulation of gene expression, cell proliferation, and differentiation (1).

FABP4 was originally identified as an adipocyte-specific protein (6), and a 5.4-kb promoter of the FABP4 gene has been widely used to target adipose tissue-specific gene expression in mice (7,8,9). Studies in FABP4-null (FABP4−/−) mice have demonstrated that it is important in the maintenance of glucose and lipid metabolism (10, 11). Thus, FABP4−/− mice are protected from development of obesity-induced insulin resistance and diet-induced atherosclerosis (12,13,14,15). Consistent with these studies, a small-molecule inhibitor of FABP4 was found to be an effective therapeutic agent in metabolic syndrome in mouse models (16). FABP4 is also detected in macrophages, where it participates in regulation of inflammatory activity and cholesterol trafficking via NF-κB and peroxisome proliferator-activated receptor (PPAR)-γ pathways (17,18,19). In other recent studies, either constitutive or induced expression of FABP4 has been found in bronchial epithelial cells (20), trophoblasts (21), and porcine coronary arterial endothelial cells (ECs) that underwent denudation and regeneration (22). Ferrell et al. (23) recently reported that FABP4 was highly expressed in lymphatics and that two mutations in the FABP4 gene in patients with primary lymphedema suggested causality.

Collectively, these recent studies suggest that FABP4 is more widely expressed than initially thought and that it may have additional biological roles in other cell types. We conducted a systematic survey of FABP4 expression in a panel of mouse and human tissues and detected intense FABP4 expression in a subset of ECs in several tissues. Expression of FABP4 in the vasculature was primarily confined to capillaries and small veins, the main angiogenic compartment of the vasculature. To begin to define the role of FABP4 in ECs, we investigated the regulation of FABP4 in ECs by proangiogenic factors, studied its effect on EC proliferation, and examined the expression of FABP4 in infantile hemangiomas, the most common tumor of ECs.

MATERIALS AND METHODS

Animal and human specimens

Adult wild-type C57BL/6J (Jackson Laboratories, Bar Harbor, ME, USA) and FABP4−/− mice on the C57BL/6J background (provided by Dr. Gokhan Hotamisligil, Harvard School of Public Health, Boston, MA, USA) (10) were used in expression studies with approval from the institutional animal care and use committees at Children’s Hospital and Harvard Medical School, Boston. Discarded surgical or autopsy human specimens were obtained from the Department of Pathology at Boston Children’s Hospital. Discarded human umbilical cord and placenta specimens were collected from Brigham and Women’s Hospital with approval from the institutional review board.

Cell culture and reagents

Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords according to the method described by Jaffe et al. (24) and cultured in M199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen), 30 μg/ml endothelial cell growth factor supplement (ECGF; BD Biosciences, Bedford, MA, USA), 1% penicillin/streptomycin (Invitrogen), and 0.1% heparin (Sigma, St. Louis, MO, USA). HUVEC starvation medium contained 2% FBS and no ECGF. Second- or third-passage HUVECs were used in all experiments. HEK-293T cells (American Type Culture Collection, Rockville, MD, USA) were cultured in DMEM (Invitrogen) supplemented with 10% FBS and 1× nonessential amino acids (Sigma). Human dermal microvascular endothelial cells (HDMEC) from newborn foreskin were isolated and cultured, as described previously (25). VEGF-A 165 (VEGF) and human basic FGF (bFGF) were purchased from R&D Systems (Minneapolis, MN, USA) and Neuromics (Edina, MN, USA), respectively. Recombinant human placental growth factors (PlGFs) 1 and 2 were from Shenandoah Biotechnology (Warwick, PA, USA). SU1498 was purchased from Calbiochem (San Diego, CA, USA). Recombinant human FABP3, FABP4, and FABP5 were from Cayman Chemical (Ann Arbor, MI, USA).

RNA interference

A set of Mission short-hairpin RNAs (shRNAs) targeting VEGFR2 kinase insert domain receptor, human FABP4, and shRNA control vector targeting firefly luciferase were purchased from Sigma. Clone IDs of these vectors are provided in Supplemental Table S2. Lentiviral shRNA transfer vectors and four expression vectors encoding viral packaging proteins (provided by Dr. Richard Mulligan, Children’s Hospital, Boston, MA, USA) were cotransfected into HEK293 cells, as described previously (26). Supernatants of HEK293T cells were collected and used to transduce HUVECs. Puromycin (2 μg/ml; Sigma) was added to the medium for 24 h for enrichment of transduced cells.

Cell proliferation

HUVECs or HDMECs were transduced with lentiviruses encoding shRNA for FABP4 or firefly luciferase and plated into 96-well plates in quadruplicate. Cells were cultured in the starvation medium with 2% FBS for 8 h. The medium was replaced with the complete medium, and cells were treated with the vehicle (0.1% DMSO), VEGF (10 ng/ml), or bFGF (10 ng/ml) for 24 h. BrdU incorporation was measured using a chemiluminescence-based cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions.

Real-time RT-PCR

Total RNA was isolated from frozen adult mouse tissues with TRIzol reagent (Invitrogen) and treated with DNase I (Invitrogen). First-strand cDNA was synthesized from 0.5 μg of RNA with the Superscript II First-Strand Synthesis System (Invitrogen) using 0.5 μg oligo-dT. Pooled first-strand cDNA samples from various human tissues were purchased from Clontech (Palo Alto, CA, USA). Real-time PCR analysis employed the Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA) and the brilliant SYBR Green QPCR Master Mix (Stratagene). PCR conditions were as follows: initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 30 s. All reactions were performed in triplicate and repeated at least 2 times. Cyclophilin A or β-actin was used as an internal reference to normalize the target transcripts by the 2−ΔΔCTmethod (27). The lowest value for each gene was assigned an arbitrary level of 1. Real-time PCR primers were designed with the IDT DNA Web site (http://www.idtdna.com; Supplemental Table S1).

Immunoblot analysis

Frozen tissue samples were homogenized in 10 vol of phosphate buffer, at pH 6.8, and supplemented with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Cells were lysed in buffer containing 0.5% Triton X-100, 100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4), Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA), and the protease inhibitor cocktail. Total protein concentration was calculated by the Bradford method. Immunoblot analysis was performed as described previously (28). All primary antibody incubations were performed at 4°C overnight at the following dilutions: monoclonal mouse anti-FABP3 (Abcam, Cambridge, MA, USA), 1:2500; polyclonal rabbit anti-FABP4 antibody (Abcam), 1:2000; polyclonal rabbit anti-FABP5 antibody (Cell Sciences, Canton, MA, USA), 1:500; mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International, Billerica, MA, USA) 1:2000; mouse monoclonal anti-VEGFR2 (Santa Cruz, Santa Cruz, CA, USA), 1:200; rabbit polyclonal anti-phospho-VEGFR2 (Tyr1175) (Cell Signaling, Beverly, MA, USA), 1:1000.

Immunohistochemistry and double immunofluorescence

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded or frozen tissue sections, as described previously (29). All primary antibody incubations were performed overnight at 4°C, and the primary antibodies were used at the following dilutions: anti-FABP4, 1:200; anti-FABP3, 1: 40; monoclonal anti-CD31 (Dako, Carpenteria, CA, USA), 1:50. Antigen retrieval was performed for CD31 with Tris-EDTA, pH 9, buffer at 95°C for 15 min. For double immunofluorescence, secondary antibodies were Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Negative controls included rabbit or mouse primary antibody isotype controls and use of anti-FABP4 antibody for FABP4−/− mouse tissues.

Statistical analysis

All results are presented as means ± sd from ≥3 independent experiments. Statistical significance was determined with the nonparametric Mann-Whitney U test (2 tailed). Values of P < 0.05 were considered significant.

RESULTS

FABP expression in mouse tissues

The relative steady-state mRNA levels of FABP4 and 2 other closely related FABPs, FABP3, and FABP5 were determined by real-time PCR (Fig. 1A). As expected, mRNA levels of FABP3 were the highest in the heart. Other tissues showed minimal (liver and brain) or no expression of FABP3 mRNA. The highest FABP4 mRNA levels were also detected in the heart, followed by kidney, liver, small intestine, and lung. Brain and spleen were the only tissues in the panel without any detectable FABP4 mRNA. FABP5 is known to be the most ubiquitously expressed FABP. Accordingly, the mRNA levels of FABP5 in various mouse tissues varied by only severalfold, compared with thousands-fold differences in the case of FABP3 and FABP4. The highest FABP5 mRNA levels were also detected in the heart followed by liver and brain, whereas the lowest FABP5 mRNA levels were detected in the kidney. These results were consistent with those obtained from a panel of pooled first-strand cDNA samples from various human tissues (not shown).

Figure 1.

Figure 1.

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FABP4 expression in mouse tissues. A) Relative mRNA expression levels of FABP3, FABP4, and FABP5 in adult mouse tissues. Pooled cDNAs from various mouse tissues (n=3) served as templates in real-time PCR assays. Relative expression levels were normalized to β-actin by the 2−ΔΔCT method. An arbitrary level of 1 was assigned to the tissue with the lowest value for each gene. B) FABP4 protein expression in adult mouse tissues was analyzed by immunoblotting with an anti-FABP4 antibody. Recombinant FABP4 (rFABP4, first lane) was used as a positive control. GAPDH was used to normalize the total protein loaded in each lane.

Next, FABP4 protein levels in adult mouse tissues were examined by immunoblotting (Fig. 1B). The specificity of the commercial polyclonal FABP4 antibody was confirmed as described in Supplemental Data (Supplemental Fig. S1). Consistent with the RT-PCR results, the highest level of FABP4 expression was detected in the heart followed by renal and the intestinal tissue. There was also a faint band in liver samples in overexposed blots (not shown).

Cellular localization of FABP4 in mouse and human tissues

To determine the cellular source of FABP4, immunohistochemistry was performed on formalin-fixed, paraffin-embedded adult mouse tissues (Fig. 2A). In the heart, FABP4 was immunolocalized to ECs of capillaries and small veins in the myocardium. There was no FABP4 immunoreactivity in arterial ECs, whereas the majority of endocardial ECs were FABP4-positive (not shown). The specificity of the FABP4 antibody was confirmed by lack of immunoreactivity on FABP4−/− mouse heart tissues. In the lung, alveolar capillary and arterial ECs were consistently negative for FABP4 expression, whereas some pulmonary venous ECs were positive. Periadventitia of extraparenchymal large airways and vessels also contained some FABP4-immunoreactive adipocytes and capillary ECs. In the kidney, FABP4 was detected in peritubular capillary ECs in both the cortex and the medulla. Notably, glomerular capillary ECs and arterial ECs demonstrated minimal or no FABP4 staining. In liver samples, FABP4 expression was observed in portal and hepatic veins and their proximal branches, while cells lining the sinusoids did not demonstrate FABP4 immunoreactivity. FABP4 was also detected in capillary ECs in the skeletal muscle and pancreas but was absent in the brain and spleen (not shown). In general, FABP4 was detected in both the cytoplasm and nucleus in most ECs.

Figure 2.

Figure 2.

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Immunolocalization of FABP4 in mouse and human tissues. A) Immunohistochemical distribution of FABP4 in mouse tissues. Images are representative (n=3–5/tissue). Myocardial microvascular and small venous ECs are immunoreactive for FABP4 in WT, but not in FABP4−/− mice (negative control). In the lung, FABP4 is detected in pulmonary venous (red arrowheads), but not arterial (black arrowheads), or alveolar capillary ECs. Periadventitial microvessels (some marked by red arrows) and adipocytes (black arrows) are also FABP4-positive. In the kidney, FABP4 is detected in peritubular microvascular, but not glomerular (G) or arterial (A) ECs. Hepatic and portal vein branches in the liver and intestinal capillaries also harbor FABP4 immunoreactive ECs. In all tissues, FABP4 expression is noted in both the cytoplasm and the nucleus of the ECs. B) FABP4 and FABP5 expression in mature placenta. FABP4 immunoreactivity in ECs is detected mostly in small vessels in tertiary and some secondary villi, but most vessels in primary villi vessels (black arrows) are negative, whereas FABP5 is expressed in a more uniform fashion in the placenta, including bigger vessel ECs of the primary villi (red arrows). C) Umbilical cord vein ECs demonstrate no immunoreactivity for FABP4 but are uniformly positive for FABP5. Methyl green was used as the counterstain. Scale bars = 50 μm.

For a comparative analysis, some normal human tissues were immunostained for FABP4. In general, the expression pattern of FABP4 in human tissues was similar to that in mouse tissues, with a few exceptions. In human liver samples, FABP4 immunoreactivity was not observed in ECs, but rather in smooth muscle cells of hepatic artery branches in the portal triad (Supplemental Fig. S2). In human lung samples, FABP4 was detected in microvessel ECs surrounding intraparenchymal large airways and vessels and was rarely present in pulmonary vein ECs (not shown). In mature placenta samples, FABP4 was detected in vascular ECs of the tertiary and some secondary villi, but was not observed in the primary villi (Fig. 2B). The placental expression pattern of FABP4 differed from that of FABP5, which was expressed in ECs in the majority of vessels, including the primary villi. The ECs of both the umbilical cord vein and artery were negative for FABP4, but positive for FABP5 (umbilical vein shown in Fig. 2C). The endothelial localization of FABP4 was confirmed by double immunofluorescence using anti-CD31 and anti-FABP4 antibodies in a human myocardium sample (Fig. 3).

Figure 3.

Figure 3.

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FABP4 is colocalized with CD31 in microvascular endothelial cells in the myocardium. Double immunofluorescence staining for CD31 and FABP4 was performed on a paraffin-embedded human myocardial tissue section. Scale bars = 50 μm.

FABP4 expression in infantile hemangioma

Infantile hemangioma, the most common tumor of infancy, is characterized by excessive proliferation of ECs in the first year of life followed by slow regression during early childhood. We examined the expression of FABP4 in paraffin-embedded infantile hemangioma samples (Fig. 4A). In proliferating hemangiomas (n=4), diffuse and intense cytoplasmic FABP4 immunoreactivity was detected in all hemangioma ECs. In addition, some nuclei were FABP4-positive. The majority of the ECs of draining veins were also positive for FABP4, whereas pericytes and some arterial ECs were negative. In involuting hemangiomas (n=4), FABP4 expression was detected in the majority of ECs. However, FABP4 expression in some large vessels was weaker than in proliferating hemangiomas. FABP4 was also detected in adipocytes (Fig. 4A, middle panel; A) in both proliferating and involuting hemangiomas. In normal skin samples, FABP4 was detected mostly in small venous and microvascular ECs. Normal skin arteries and some small veins were negative for FABP4 immunoreactivity. In addition to ECs, FABP4 was expressed in some melanocytes (Supplemental Fig. S3) and, as expected, in subcutaneous adipocytes. Double immunofluorescence with FABP4 and CD31 antibodies confirmed the endothelial localization of FABP4 in proliferating (Fig. 4B) and involuting hemangiomas (Fig. 4C).

Figure 4.

Figure 4.

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FABP4 is expressed in infantile hemangioma endothelial cells. A) FABP4 is expressed in ECs of both proliferating and involuting hemangiomas. In normal-appearing adjacent skin samples, FABP4 is expressed in small veins and microvessels (red arrows). B) Double immunofluorescence for FABP4 (green) and CD-31 (red) demonstrates coexpression of these proteins in proliferating hemangioma ECs, including ECs of large intralesional vessels (white arrow). C) FABP4 (green) and CD31 (red) are colocalized in most involuting hemangioma ECs, but most large-vessel ECs are negative for FABP4 immunoreactivity (white arrowhead). Scale bars = 100 μm.

Regulation of FABP4 in endothelial cells by VEGF and bFGF

To determine whether FABP4 expression was regulated by VEGF, the steady-state relative mRNA levels of FABP4 in response to VEGF treatment over time were examined by real-time PCR. FABP4 mRNA levels remained stable at 3 and 6 h following VEGF stimulation but were significantly increased by 2.6-, 2.8-, and 3.2-fold at 9, 12, and 24 h, respectively (Fig. 5A). At the protein level, FABP4 was detected as a 15-kDa band in unstimulated HUVEC lysates (please note that the signal varied with different exposure times in different experiments), and FABP4 levels significantly increased by 5.2- and 6.5-fold in response to 10 and 50 ng/ml VEGF, respectively (Fig. 5B). bFGF treatment did not affect FABP4 mRNA levels up to 12 h, but there was a 3-fold increase at 24 h (P<0.05, Fig. 4C). There were also significant increases in FABP4 protein levels in response to bFGF in HUVECs (Fig. 5D). FABP4 mRNA and protein showed similar alterations in HDMECs stimulated with VEGF and bFGF (not shown).

Figure 5.

Figure 5.

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FABP4 expression is induced by VEGF and bFGF. A, C) HUVECs were starved in ECGF-free M199 with 2% FBS for 8 h, then stimulated with 50 ng/ml VEGFA165 (A) or 10 ng/ml bFGF (C). Cells were harvested at the indicated time points, and FABP4 mRNA expression was analyzed by real-time PCR. *P < 0.05 vs. 0 h. B, D) HUVECs were stimulated with the indicated dose of VEGFA165 (B) or bFGF (D) for 48 h, and FABP4 protein expression was analyzed by immunoblotting and densitometry. *P < 0.05 vs. vehicle control.

VEGF binds and activates two distinct receptor tyrosine kinases, VEGF-receptor-1 (VEGFR-1/Flt1), and VEGFR-2 (mouse Flk-1/human KDR), leading to the recruitment and subsequent phosphorylation of adaptor proteins and other downstream effectors (30). The effects of selective VEGFR-1 ligands, PlGF1 and PlGF2, were tested to determine whether FABP4 expression is mediated by VEGFR-1 activation (31). There were no significant differences in FABP4 mRNA (not shown) or protein levels between control and PlGF1- or PlGF2-treated cells (Fig. 6A). In parallel experiments, the effect of the VEGFR-2-selective inhibitor SU1498 (32,33,34) on VEGF-induced FABP4 expression was determined. HUVECs that were pretreated with SU1498 for 1 h before VEGF stimulation showed a significant decrease in FABP4 levels compared with the controls (Fig. 6B), suggesting that VEGFR-2 signaling mediates the induction of FABP4. To confirm these results, HUVECs were transduced with lentiviruses encoding 3 different shRNAs targeting VEGFR2 (shRNA1-3) and one targeting firefly luciferase as a negative control. The efficiency of shRNAs was determined by examining the levels of phosphorylated and total VEGFR2 in response to VEGF treatment for 10 min and 24 h, respectively. Both phosphorylated and total VEGFR2 levels were significantly lower in HUVECs that were transduced with shRNA2 and shRNA3 than with shRNA1 and firefly luciferase shRNA (Fig. 6C). Next, HUVECs were transduced with shRNAs and treated with VEGF or bFGF for 24 h, and cell lysates were analyzed for FABP4 expression. FABP4 levels were significantly lower in VEGF-stimulated HUVECs transduced with shRNA2 and shRNA3 than with the control shRNAs. VEGFR2 shRNA treatment did not result in any differences in FABP4 levels in bFGF-stimulated HUVECs. Collectively, these results indicate that VEGF induces FABP4 expression primarily through the activation of the VEGFR-2 receptor in ECs, whereas the effect of bFGF is VEGFR-2 independent.

Figure 6.

Figure 6.

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FABP4 expression is induced by VEGF via VEGFR2. A) HUVECs were treated with PlGF1 or PlGF2 for 48 h, and FABP4 expression was analyzed by immunoblotting. B) HUVECs were pretreated with the VEGFR2 inhibitor SU1498 at the indicated doses for 1 h, then with VEGF for 48 h, and FABP4 expression was assessed. C) HUVECs were transduced with shRNAs targeted against VEGFR2 (shRNA1, shRNA2, and shRNA3) or firefly luciferase (control shRNA), treated with puromycin for 24 h and then with VEGF for 10 min or 24 h, and phosphorylated VEGFR2 (pVEGFR2) and total VEGFR2 levels were assessed. D) FABP4 expression was analyzed in VEGFR2-knockdown and control HUVECs after VEGF (50 ng/ml) or bFGF (10 ng/ml) treatment for 24 h.

Effect of FABP4 on endothelial cell proliferation

Up-regulation of FABP4 expression by VEGF and bFGF suggested that it could play a role in EC proliferation. To investigate this hypothesis, HUVECs were transduced with lentiviruses encoding a control shRNA (c-shRNA) or two different shRNAs (shRNA1 and shRNA2) targeting FABP4. The effect of these shRNAs on FABP4 protein expression was analyzed by immunoblotting (Fig. 7A). FABP4 protein levels were significantly reduced in HUVECs that were transduced with either shRNA1 or shRNA2, whereas no changes were observed in HUVECs that were transduced with the c-shRNA. Next, cell proliferation was assessed by BrdU incorporation. Cell proliferation was significantly decreased under both baseline conditions and in response to VEGF or bFGF in FABP4-knockdown HUVECs as compared with the c-shRNA-transduced cells (Fig. 7B). Similar results were obtained in cells that were transduced with the FABP4-shRNA2 and also in HDMECs (not shown).

Figure 7.

Figure 7.

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FABP4 deficiency inhibits endothelial cell proliferation. A) HUVECs were transduced with shRNA targeting the firefly luciferase (C) or FABP4 (1 and 2) and treated with puromycin for 24 h. Cells were harvested 1, 3, and 5 d after the puromycin treatment, and FABP4 expression was analyzed by immunoblotting for FABP4. β-Actin was used as a loading control. B) HUVECs were transduced with the control shRNA or FABP4-shRNA1. Cells were treated with the vehicle (0.1% DMSO), VEGF (10 ng/ml), or bFGF (10 ng/ml) for 24 h. Cell proliferation was measured by BrdU incorporation using an ELISA kit.

Microvessel density in FABP4−/− mice

To determine whether FABP4 has an effect on microvessel development or survival under physiological conditions, the microvessel density in the myocardium of WT and FABP4−/− mice was examined. There were no statistically significant differences in the density of CD31-positive microvessel ECs between WT and FABP4−/− myocardial tissues (Supplemental Fig. S4).

DISCUSSION

FABP4 has been regarded as a primarily adipocyte and macrophage-specific intracellular lipid-binding protein. Herein, we show that FABP4 is also expressed in microvascular ECs of several mouse and human tissues. The tissue-specific expression pattern of FABP4 in capillary ECs does not appear to correlate with the phenotypic features of ECs. For example, ECs in both the myocardium and the brain are continuous and nonfenestrated (35, 36), but FABP4 is expressed in the ECs of myocardium, and not those in the brain. Pulmonary microvasculature is another exception to the widespread expression of FABP4 in capillary ECs; whereas FABP4 was not detected in alveolar capillaries, it was present in microvascular ECs surrounding large airways and vessels. These latter structures are derived from the bronchial circulation, which has a greater capacity for angiogenesis than ECs from the pulmonary circulation (36,37,38). This expression pattern suggests that FABP4 may be a useful marker to discriminate between the two microcirculations in the lung, a feature that is not shared by any other EC marker described to date.

Previous reports have indicated the expression of two other members of the FABP family, FABP3 and FABP5, in ECs (39, 40). Our data showed that FABP3 mRNA is primarily expressed in the heart, and abundant FABP3 protein is detected in cardiomyocytes, but not in myocardial vessels (Supplemental Fig. S5). Furthermore, in contrast to a previous report, we detected FABP5 in microvascular, as well as nonmicrovascular vessels, such as the umbilical vessels (40). These discrepancies may be due to different sensitivities of the antibodies used in these studies.

FABP4 was expressed in an angiogenesis-dependent pathology, infantile hemangioma, being the most common tumor of infancy and ECs (41). Infantile hemangiomas proliferate rapidly in infancy and involute in early childhood. Hemangioma ECs are characterized by increased VEGFR2-dependent signal transduction (42). The proliferating phase is characterized by cellular masses without a well-defined vascular architecture, whereas the proliferation slows and the vascular architecture becomes more defined in the involuting phase (43). There was consistent and intense expression of FABP4 in both the proliferating and involuting hemangioma ECs. One difference between the two stages was the vanishing expression of FABP4 from larger vessel ECs in involuting hemangiomas. During this stage, blood vessels regress, and an adipocyte-rich connective tissue accumulates. Adipocytes express large quantities of FABP4 and are found in both normal subcutaneous tissue and involuting hemangiomas. Therefore, whole-tissue microarray expression analysis would not have revealed the abundant expression of FABP4 in hemangioma ECs (44). Interestingly, a placental origin has been proposed for the hemangioma ECs (45), and our finding that FABP4 is expressed in both placental vessel ECs and hemangioma ECs is consistent with this hypothesis.

To begin to understand the functional significance of FABP4 expression in ECs, we first examined its relationship with VEGF, the key regulator of angiogenesis in both physiological and pathological conditions, and bFGF, another important EC mitogen. Recent studies have suggested that VEGF also acts as a survival factor for normal microvasculature, as inhibition of VEGF results in significant capillary regression in several mouse organs, mostly with fenestrated ECs (46, 47). FABP4 mRNA was significantly increased 9 h after VEGF treatment, consistent with a direct response. In contrast, there was no change in FABP4 mRNA levels at 9 h and 12 h following bFGF treatment, but there was a significant increase at 24 h. VEGF also led to a strong and dose-dependent induction of FABP4 protein expression, whereas bFGF induced a significant, but less robust increase in FABP4 protein levels. Although HUVECs were used as the primary cell culture system, these results were verified in HDMECs. Interestingly, in contrast to their robust expression of FABP4 in vitro, no expression was detected in the umbilical cord vein ECs in situ. This finding is consistent with the absence of FABP4 expression in most large-vessel ECs, as well as the association between FABP4 expression and EC proliferation.

VEGF induces FABP4 via VEGFR2 and not VEGFR1; treatment with the VEGFR1 agonists PlGF1 and PlGF2 did not increase FABP4 expression, whereas chemical inhibition and shRNA-mediated knockdown of VEGFR2 almost completely inhibited the induction of FABP4 by VEGF. It is well known that the angiogenic activities of VEGF, including EC proliferation, survival, migration, and permeability, are transduced mainly through VEGFR2. Thus, our data suggested that FABP4 could have a role in the angiogenic activities mediated by the VEGF/VEGFR2 pathway. Indeed, knockdown of FABP4 dramatically reduced proliferation of ECs both under baseline conditions and in response to VEGF and bFGF in vitro. FABP4 is normally expressed in some microvascular ECs, but we did not find a difference in the density of myocardial microvessels between FABP4−/− and WT mice. This finding indicates that FABP4 is not required for microvessel formation or survival under physiological conditions in vivo in mice. However, further in vivo studies will be required to determine whether FABP4 promotes survival or proliferation of microvessel ECs in pathological conditions characterized by activation of the VEGF/VEGFR2 pathway, such as ischemia, hypoxia, or tumor development.

Other members of the FABP family have been implicated in cell proliferation and migration in both normal and cancer cells. For example, down-regulation of FABP7 significantly reduces proliferation of melanoma cells and neurons in cultured rat embryo brains (48,49,50), and suppression of FABP5 impairs the motility of keratinocytes during wound healing (51). In another recent study, FABP5 promoted cell survival in response to retinoic acid via signaling through PPAR-β (52). Unlike FABP5, FABP4 interacts with PPARγ in several cell types, including macrophages (53,54,55). PPARγ and PPARβ are both expressed in ECs, where they might be responsible for opposing effects on EC survival and proliferation (56,57,58). Our tissue expression data showing both nuclear and cytoplasmic expression of FABP4 in ECs support previous in vitro studies (3), and the possibility that FABP4 may interact with a nuclear target, such as PPARγ, in ECs.

Collectively, our findings demonstrate that FABP4 is a useful marker for infantile hemangioma ECs, as well as microvascular ECs in some tissues. FABP4 is a target of the VEGF/VEGFR2 pathway and a positive regulator of EC proliferation in vitro. These findings suggest that FABP4 may have a novel role in postnatal angiogenesis.

Supplementary Material

Supplemental Data
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Acknowledgments

This study was supported in part by the Peabody Foundation, Inc. (S.C.) and U. S. National Institutes of Health grant 5T32 HD007466-12 (Y.T.). We are grateful to Dr. Gokhan Hotamisligil (Harvard School of Public Health, Boston, MA, USA) for the FABP4−/− mice and valuable discussions, and Drs. Patricia D'Amore and Stella Kourembanas for critical review of the manuscript. We also thank Drs. Alessia Di Nardo and Mustafa Sahin for the shRNA protocol. We are grateful to Marcia Filip, RN (Center for Clinical Investigation, Brigham and Women’s Hospital, Boston, MA, USA), for her help with umbilical cord collection.

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