Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. (A) HUVECs were incubated with S1P for 4 h at the indicated concentrations. RNAs were harvested and analyzed by real-time PCR using specific primer sets for human VEGF-C or GAPDH. (B) HUVECs were incubated with S1P (5 μmol/L) for different time intervals as indicated and subjected to real-time PCR. (C) VEGF-C levels in the supernatant of cultured HUVECs were measured by ELISA after incubation with S1P for 8 h at various concentrations as indicated. (D) HUVECs were incubated with S1P (5 μmol/L) for the indicated time intervals and VEGF-C levels in the supernatant were measured by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression by a matrix metalloproteinase (MMP)-2-dependent pathway in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were pretreated with GM6001 (10 μmol/L) for 1 h, followed by S1P (5 μmol/L) treatment for another 4 h. VEGF-C mRNA expression levels were monitored by real-time PCR. (B) siRNA mediated downregulation of MMP-2 mRNA expression in HUVECs. MMP-2 and scrambled siRNA were transfected into HUVECs by electroporation. The transfection efficiency was evaluated by real-time PCR. MMP-2 expression was suppressed in HUVECs transiently transfected with MMP-2 siRNA. (C) HUVECs transfected with scrambled or MMP-2 siRNA were incubated with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. (D) HUVECs were incubated with S1P (5 μmol/L) for 8 h, and the secreted concentration of VEGF-C protein was determined by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Electroporation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression by a fibroblast growth factor receptor (FGFR)-1-dependent pathway in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were pretreated with SU5402 (5 μmol/L) or AG1478 (100 nmol/L) for 1 h, followed by S1P (5 μmol/L) treatment for another 4 h. VEGF-C mRNA expression levels were monitored by real-time PCR. (B) Total mRNA was extracted from HUVECs, and reverse-transcribed to cDNA. According to real-time PCR, the relative levels of different FGF receptors normalized to GAPDH are shown. (C) FGFR-1 and scrambled siRNA were transfected into HUVECs by electroporation. The transfection efficiency was evaluated by real-time PCR. FGFR-1 expression was suppressed in HUVECs transiently transfected with FGFR-1 siRNA. (D) HUVECs were transfected with scrambled or FGFR-1 siRNA, followed by incubation with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. (E) HUVECs were incubated with S1P (5 μmol/L) for 8 h, and the secreted concentration of VEGF-C protein was determined by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control. fP<0.01 vs S1P.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription, Transfection, Electroporation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) induces fibroblast growth factor (FGF) receptor (FGFR)-1 phosphorylation in a time-dependent manner in human umbilical vein endothelial cells (HUVECs). (A) Starved HUVECs were treated with S1P (5 μmol/L) for 1 and 5 min. The concentration of phosphorylated FGFR-1 was measured by an FGFR-1-phospho ELISA kit. (B) HUVECs were pretreated with GM6001 (5 μmol/L) for 1 h, followed by S1P or FGF-2 treatment for 1 min. The concentration of phosphorylated FGFR-1 was measured by an FGFR-1-phospho ELISA kit. All ELISA data are expressed as the mean±SD from at least three independent experiments. bP<0.05 vs control. eP<0.05 vs S1P.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Phospho-proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P)-upregulated vascular endothelial growth factor (VEGF)-C mRNA expression is suppressed by shRNA of fibroblast growth factor (FGF)-1 but not siRNA of FGF-2 in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with scrambled, FGF-1 shRNA (A), and FGF-2 siRNA (B) by electroporation, and the transfection efficiency was evaluated by real-time PCR. FGF-1 (C) and FGF-2 knockdown (D) HUVECs were incubated with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, shRNA, Transfection, Electroporation, Real-time Polymerase Chain Reaction, Knockdown, Incubation, Control