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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.