Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ectopic Fatty Acid–Binding Protein 4 Expression in the Vascular Endothelium is Involved in Neointima Formation After Vascular Injury

doi: 10.1161/JAHA.117.006377

Figure Lengend Snippet: Effects of the conditioned medium of Fabp4 ‐overexpressed endothelial cells in vascular smooth muscle cells. A, Experimental design of human coronary artery smooth muscle cells ( HCASMCs ) treated with the conditioned medium ( CM ) prepared by 24‐hour incubation of adenovirus vector of fatty acid–binding protein 4 (Ad‐ FABP 4)– and empty sequence (Ad‐Control)–transfected human coronary artery endothelial cells ( HCAECs ) in the absence and presence of 10 μg/mL anti‐ FABP 4 antibody ( FABP 4‐Ab) for 24 hours ( HCASMC ‐ CM ‐Ab). B, Gene expression of inflammatory cytokines and proliferation‐ and adhesion‐related molecules determined by quantitative real‐time polymerase chain reaction (PCR) in HCASMC ‐ CM ‐Ab (n=6 in each group). * P <0.05 vs CM of Ad‐Control–transfected HCAECs ( CM ‐Ad‐Control). † P <0.05 vs CM of Ad‐ FABP 4–transfected HCAECs ( CM ‐Ad‐ FABP 4). C and D, Proliferation of HCASMC ‐ CM supplemented with 5% FBS in the absence and presence of 10 μg/mL FABP 4‐Ab for 24 hours assessed by MTS (C) and bromodeoxyuridine (BrdU; D) assays (n=6 in each group). * P <0.05 vs CM ‐Ad‐Control. † P <0.05 vs CM ‐Ad‐ FABP 4. E, Migration of HCASMC ‐ CM supplemented with 0.5% BSA in the absence and presence of 10 μg/mL FABP 4‐Ab for 15 hours assessed by scratch wound assay (n=6 in each group). * P <0.05 vs CM ‐Ad‐Control. † P <0.05 vs CM ‐Ad‐ FABP 4. F, Experimental design of HCASMCs coincubated with Ad‐ FABP 4– and Ad‐Control–transfected HCAECs using insert transparent wells for 24 hours ( HCASMC ‐ TW ). G and H, Gene expression of inflammatory cytokines (G) and proliferation‐ and adhesion‐related molecules (H) determined by quantitative real‐time PCR in HCASMC ‐ TW (n=6 in each group). * P <0.05 vs coincubation using transparent wells with Ad‐Control–overexpressed HCAECs ( TW ‐Ad‐Control). AU indicates arbitrary unit; IL, interleukin; Itga5, integrin a 5; Itgb3, integrin b 3; Mcp1, monocyte chemotactic protein‐1; Pdgfra, platelet‐derived growth factor receptor α; Pdgfrb, platelet‐derived growth factor receptor β; Tnfa, tumor necrosis factor α.

Article Snippet: 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.

Techniques: Incubation, Plasmid Preparation, Binding Assay, Sequencing, Transfection, Expressing, Real-time Polymerase Chain Reaction, Migration, Scratch Wound Assay Assay, Derivative Assay