Fig 1: Schematic diagram of the mechanisms of pigment epithelial-derived factor (PEDF) regulation on vascular endothelial growth factor B (VEGFB) signaling affect endothelium-mediated fatty acid (FA) uptake in normal state and in condition of hyperlipidemia. EC indicates endothelial cell; FATP3/4, FA transport proteins 3 and 4; VEGFR1/Nrp-1, vascular endothelial growth factor receptor 1 and the coreceptor neuropilin 1.
Fig 2: Pigment epithelial-derived factor (PEDF) inhibits expression of vascular endothelial growth factor B (VEGFB)–targeted receptors in endothelial cells. Representative images of heart sections visualized with anti–vascular endothelial growth factor receptor 1 (VEGFR1) (A) or anti–neuropilin 1 (NRP1) (B) antibody and colocalized with CD31 (endothelial marker) antibody from wild-type (WT), apolipoprotein E–deficient (ApoE-/-), and ApoE-/-/PEDF-deficient (PEDF -/-) mice fed high-fat diet (HFD) for 36 weeks and age-matched WT fed chow diet (CD) (n=3–4 per genotype). Merged images appear as yellow. C, Representative immunofluorescent images of human umbilical vein endothelial cells (HUVECs) treated with mock (unstimulated), 100 nmol/L PEDF alone, 100 ng/mL VEGFB alone, 100 ng/mL VEGFB+100 nmol/L PEDF, and 100 ng/mL VEGFB+100 nmol/L PEDF+PEDF neutralizing antibody (PEDF-Ab) for 24 hours. D, Cultures were triple stained for VEGFR1 (red) or NRP1 (red) with CD31 (green) (magnification ×20; bar=50 µm). E, Protein expression of VEGFR1, NRP1, fatty acid transport protein (FATP) 3, and FATP4 in HUVECs after treatments with mock, increasing concentrations of VEGFB (25, 50, and 100 ng/mL), 100 ng/mL VEGFB+100 nmol/L PEDF, and 100 ng/mL VEGFB+100 nmol/L PEDF+PEDF-Ab for 24 hours (n=4). F, 4,4-Difluoro-3a,4adiaza-s-indacene—fatty acid uptake into HUVECs after the same treatments as above (n=3). Data are presented as mean±SE. DAPI indicates 4',6-diamidino-2-phenylindole; PECAM, platelet endothelial cell adhesion molecule 1. *P<0.05, ***P<0.001.
Fig 3: Vascular endothelial growth factor B signaling pathway mediating endothelial fatty acid (FA) uptake is activated in high-fat diet (HFD)–fed mice lacking pigment epithelial-derived factor (PEDF) expression. A, Representative images show 4,4-difluoro-3a,4adiaza-s-indacene–FA uptake in wild-type (WT), apolipoprotein E deficient (ApoE-/-), and ApoE-/-/PEDF-deficient (PEDF -/-) mice fed HFD for 36 weeks (1, WT mice; 2, ApoE-/- mice; 3, ApoE-/-/PEDF -/- mice; n=3). B, Relative RNA expression of endothelium-mediated FA transporter genes of FA translocase (cd36), peroxisome proliferator-activated receptor ? (ppar-?), fatty acid transport protein 3 and 4 (fatp3/4), vascular endothelial growth factor receptor 1 (vegfr1), and neuropilin 1 (nrp1) in the hearts from WT, ApoE-/-, and ApoE-/-/PEDF -/- mice fed HFD for 24 weeks and age-matched WT mice fed chow diet (CD) (n=4–5 per genotype). C, Immunohistochemical localization of FATP4. D, FATP3 in endothelial cells in the heart sections from WT, ApoE-/-, and ApoE-/-/PEDF -/- mice fed HFD for 36 weeks and age-matched WT mice fed CD (n=3–4 per genotype) (bar=50 µm). Data are presented as mean±SE. *P<0.05, **P<0.01, ***P<0.001 vs WT CD; # P<0.05, ## P<0.01, ### P<0.001 vs WT HFD; & P<0.05, && P<0.01 vs ApoE-/- HFD.
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