Journal: Circulation research

Article Title: Angiogenic Mechanisms of Human CD34 + Stem Cell Exosomes in the Repair of Ischemic Hindlimb

doi: 10.1161/CIRCRESAHA.116.310557

Figure Lengend Snippet: Uptake and transfer of CD34Exo and exosomal miRNAs by endothelial cells in vitro and in vivo. Flow cytometry analysis of CD34+ cells (A) or exosomes isolated from CD34+ cells (B), transfected as indicated; confocal image of HUVECs treated with Cy3miRNA-CD34Exo (C); flow cytometry analysis of single cell suspensions from post-ischemic hindlimb tissue injected with R-PE-CD34Exo at 2h (D); % of cells uptaking exosomes (PE) from total number of each cell types quantified from Figure 5D (E), (n=3–5, *p<0.05).

Article Snippet: Human umbilical-vein endothelial cells (HUVECs) (Lonza Inc. Allendale, NJ) were maintained in endothelial growth medium-2 (EGM ™ -2; Lonza Inc.) and starved in EBM-2 medium containing 0.25% fetal bovine serum for 24 hours before cell assays were performed.

Techniques: In Vitro, In Vivo, Flow Cytometry, Isolation, Transfection, Injection

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: Overexpression and secretion of fatty acid–binding protein 4 (FABP4) in vascular endothelial cells. A and B, Gene (A) and protein (B) expression levels of FABP 4 in human coronary artery endothelial cells ( HCAECs ) treated with 50 ng/mL of vascular endothelial growth factor ( VEGF ) for 24 hours or 100 μmol/L H 2 O 2 for 1 hour followed by 23‐hour incubation in normal culture media after washing were determined by quantitative real‐time polymerase chain reaction (n=3 in each group) and Western blot analysis, respectively. * P <0.05 vs control. C, Experimental design of adenovirus‐mediated overexpression in HCAECs using adenovirus vector of FABP 4 (Ad‐ FABP 4) or empty sequence (Ad‐Control) ( HCAEC ‐ OE ). D, Gene expression of FABP 4 in HCAEC ‐ OE (n=6 in each group). * P <0.05 vs Ad‐Control. E, Representative Western blot analysis of FABP 4 in HCAEC ‐ OE . F, Western blot analysis of nitric oxide synthase ( NOS 3) and phosphorylated NOS 3 ( pS 1177) in HCAEC ‐ OE treated with 0.5 μmol/L insulin or 50 ng/mL VEGF for 0.5 hours (n=3 in each group). * P <0.05. G and H, Gene expression of inflammatory cytokines (G) and adhesion‐related molecules (H) in HCAEC ‐ OE (n=6 in each group). * P <0.05 vs Ad‐Control. I, Western blot analysis of FABP 4 (exposure: light, L; dark, D) and GAPDH was performed using the cell lysate ( CL ) and conditioned medium ( CM ) of HCAECs supplemented with 0.5% BSA in the absence and presence of 10 μmol/L isoproterenol for 6 to 24 hours (n=3 in each group). * P <0.05. J, Secretion of FABP 4 for 24 hours in HCAEC ‐ OE measured using an enzyme‐linked immunosorbent assay kit (n=3 in each group). Values were normalized to total protein concentration of the cell lysate. * P <0.05 vs Ad‐Control. AU indicates arbitrary unit; Icam1, including intracellular adhesion; IL, interleukin; Itga5, integrin a 5; Itgb3, integrin b 3; Mcp1, monocyte chemotactic protein‐1; Sele, selectin E; Tnfa, tumor necrosis factor α.

Article Snippet: Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Lonza.

Techniques: Over Expression, Binding Assay, Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Sequencing, Enzyme-linked Immunosorbent Assay, Protein Concentration

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: Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Lonza.

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

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 endothelial cells. A, Experimental design of human coronary artery endothelial cells ( HCAECs ) 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 HCAECs ( HCAEC ‐ CM ) in the absence and presence of 10 μg/mL anti‐ FABP 4 antibody ( FABP 4‐Ab) for 24 hours ( HCAEC ‐ CM ‐Ab). B, Gene expression of inflammatory cytokines and adhesion‐related molecules determined by quantitative real‐time polymerase chain reaction (PCR) in HCAEC ‐ CM (n=6 in each group). * P <0.05 vs CM of Ad‐Control–overexpressed HCAECs ( CM ‐Ad‐Control). C, Gene expression of inflammatory cytokines and adhesion‐related molecules determined by quantitative real‐time PCR in HCAEC ‐ CM ‐Ab (n=4 in each group). * P <0.05 vs FABP 4‐Ab (−). D, Experimental design of HCAECs coincubated with Ad‐ FABP 4– and Ad‐Control–transfected HCAECs using insert transparent wells for 24 hours ( HCAEC ‐ TW ). E and F, Gene expression of inflammatory cytokines (E) and adhesion‐related molecules (F) determined by quantitative real‐time PCR in HCAEC ‐ 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; Icam1, including intracellular adhesion; IL, interleukin; Itga5, integrin a 5; Itgb3, integrin b 3; Mcp1, monocyte chemotactic protein‐1; Sele, selectin E; Tnfa, tumor necrosis factor α.

Article Snippet: Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Lonza.

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

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 exogenous fatty acid–binding protein 4 (FABP4) treatment in vascular endothelial cells. A, Experimental design of human coronary artery endothelial cells ( HCAECs ) treated with 200 nmol/L recombinant FABP 4 ( HCAEC ‐Rec) in the absence and presence of 10 μg/mL anti‐ FABP 4 antibody ( FABP 4‐Ab) for 24 hours ( HCAEC ‐Rec‐Ab). B, Gene expression of inflammatory cytokines and adhesion‐related molecules determined by quantitative real‐time polymerase chain reaction (PCR) in HCAEC ‐Rec (n=3 in each group). * P <0.05 vs Rec‐ FABP 4 (−). C, Gene expression of inflammatory cytokines and adhesion‐related molecules determined by quantitative real‐time PCR in HCAEC ‐Rec‐Ab (n=3 in each group). * P <0.05 vs FABP 4‐Ab (−). D, Representative Western blot analysis of endogenous FABP 4, His‐tagged FABP 4 (His‐ FABP 4), and GAPDH was performed using the cell lysate ( CL ) and conditioned medium ( CM) of HCAECs treated with 0 to 200 nmol/L His‐tagged Rec‐ FABP 4 for 6 hours. E, Western blot analysis of nitric oxide synthase 3 ( NOS 3) phosphorylated NOS 3 ( pS 1177) in HCAEC ‐Rec‐Ab followed by stimulation with 50 ng/mL vascular endothelial growth factor ( VEGF ) for 0.5 hours (n=3 in each group). * P <0.05. AU indicates arbitrary unit; Icam1, including intracellular adhesion; IL, interleukin; Itga5, integrin a 5; Itgb3, integrin b 3; Mcp1, monocyte chemotactic protein‐1; Sele, selectin E; Tnfa, tumor necrosis factor α.

Article Snippet: Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Lonza.

Techniques: Binding Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Western Blot