Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 2. PAR-2–induced sVEGFR-1 release from endothelial cells depends on PKC activity. A, HUVECs and (B) HEK293 cells trans- fected with a VEGFR-1 promoter luciferase reporter construct were pretreated with the PKC inhibitor (Ro-32-0432; 1 mol/L) for 45 minutes and then stimulated with PAR-2 activating peptide (10 mol/L of 2f-LIGRLO-NH2) for 24 hours, and the cell culture superna- tants were assayed for sVEGFR-1 by ELISA (A) and cell lysates assayed for luciferase activity (B), respectively. C, HUVECs were infected overnight with 100 ifu per cell of adenoviruses expressing dominant-negative PKC (dnPKC), PKC (dnPKC), PKC (dnPKC), or empty vector (EV), incubated for 24 hours, and the expression of PKC isoforms were examined in cell lysates by Western blotting. These cells were stimulated with PAR-2–activating peptide (10 mol/L of 2f-LIGRLO-NH2) for 24 hours, and the conditioned medium was assayed for sVEGFR-1 by ELISA. D, PKC-1 siRNA was introduced into HUVECs using an Amaxa nucleofector and the knockdown of PKC1 confirmed by Western blotting. PAR-2–mediated sVEGFR-1 release was inhibited in cells treated with PKC-1 siRNA. Results represent the mean (SEM). *P0.01 (A and B vs control and C vs EV2f-LIGRLO); *P0.05 (D vs control).

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Activity Assay, Luciferase, Construct, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Incubation, Western Blot, Knockdown, Control

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 4. ERK-1/2 activation is required for PAR-2–induced sVEGFR-1 release. A, HUVECs and (B) HEK293 cells transfected with a VEGFR-1 promoter luciferase reporter construct were pretreated with an MEK-1/2 inhibitor (U0126; 10 mol/L) for 45 minutes, stimu- lated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) for 24 hours, and the conditioned medium was assayed for sVEGFR-1 by ELISA (A) and luciferase activity determined in cell lysates (B), respectively. C through G, Western blot analysis: cell lysates were immunoblotted for phosphor–ERK-1/2 (p-ERK), phosphor-Src (pY416Src), and phospho–Raf-1 (pS338Raf-1); HUVECs were either pre- incubated for 45 minutes with (C) MEK-1/2 (U0126; 10 mol/L), (E) Src (PP2; 10 mol/L), and (G) PKC (GF109203X; 5 mol/L) inhibitors or (D and F) infected overnight with adenoviruses expressing dominant-negative PKC (dnPKC), PKC (dnPKC), or PKC (dnPKC) and stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) for 10 minutes. *P0.01 (A) and *P0.05 (B) vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Activation Assay, Transfection, Luciferase, Construct, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Incubation, Infection, Expressing, Dominant Negative Mutation, Control

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 5. EGFR transactivation is required for PAR-2–induced sVEGFR-1 release. A, HUVECs were pretreated for 45 minutes with AG1478 (3 mol/L), stimulated for 10 minutes with PAR-2–activating peptides (2f-LIGRLO-NH2; 10 mol/L) or EGF (50 ng/mL), and the level of phosphorylated and total EGFRs were determined in cell lysates (100 g per well) by ELISA (R&D Systems). B, HUVECs were pretreated with AG1478 and stimulated for 24 hours with PAR-2–activating peptide (2f-LIGRLO-NH2), FXa (200 nmol/L), or EGF (50 ng/mL), and sVEGFR-1 was assayed in the cell culture supernatants by ELISA. C, After pretreatment with the EGFR inhibitor (AG1478), HUVECs were incubated with 2f-LIGRLO-NH2 and FXa (200 nmol/L) for 10 minutes, and cell lysates (30 g per lane) were subjected to Western blotting for phosphor-ERK1/2 (p-ERK) and -actin as a loading control. D, HUVECs were incubated with the Src inhibitor PP2 for 45 minutes and then stimulated for 10 minutes with 2f-LIGRLO-NH2, and the levels of EGFR and phosphorylated EGFR were deter- mined in cell lysates (100 g per well) by ELISA. E, HUVECs were treated with AG1478 before incubation with 2f-LIGRLO-NH2 for 10 minutes, and cell lysates were Western blotted for phosphorylated-Src (p-Src). F, HUVECs were preincubated with the metalloprotein- ase inhibitor (GM6001; 10 mol/L) or control (rGM6001; 10 mol/L) for 45 minutes before stimulation with 2f-LIGRLO-NH2 or FXa (200 nmol/L), and sVEGFR-1 was assayed in cell supernatants by ELISA. Results represent the mean (SEM); n3. *P0.05 (A and D) vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Western Blot, Control

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 6. PAR-2-induced sVEGFR-1 release is inhibited by statins, HO-1, and CO. A, HUVECs were stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) or FXa (200 nmol/L) in the presence or absence of Simvastatin (10 mol/L) for 24 hours, and sVEGFR-1 was quantified in cell supernatants by ELISA. HO-1 upregulation in HUVECs incubated with Simvastatin (10 mol/L) for 24 hours was confirmed by Western blotting (inset). B, PAEC-PAR-2 and (C) HEK-293 cells were transfected with the VEGFR-1 promoter- reporter construct, stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2) for 24 hours in the presence or absence of Simvastatin (10 mol/L), and luciferase activity was determined in cell lysates. D, HUVECs were then infected with 50 ifu per cell of HO-1 or control (EV) adenovirus for 48 hours before stimulation with 2f-LIGRLO-NH2 for 24 hours (*P0.001 vs control). HUVECs infected with low (5 ifu per cell; lanes 2 and 4) or high (50 ifu per cell; lanes 3 and 5) concentrations of HO-1 adenovirus for 24 (lanes 2 and 3) or 48 (lanes 4 and 5) hours or uninfected control cells (lane 1) were immunoblotted for HO-1 (inset). E, HEK293 cells containing the VEGFR-1 promoter-reporter construct were electroporated with HO-1 siRNA and incubated overnight. After stimulation with 2f-LIGRLO-NH2 (10 mol/L) for 24 hours, luciferase activity was determined in cell lysates. The medium was then collected and assayed for sVEGFR-1 by ELISA. F, HUVECs were treated with 50 mol/L of CORM-2 or CORM-2 control for 45 minutes and then stimulated for 24 hours with 2f-LIGRLO-NH2. Results represents mean (SEM); n3. A through C, *P0.01 vs control; D, *P0.001 vs control; **P0.001 vs EV2f-LIGRLO; E, *P0.05, *P0.001, and **P0.05 vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Transfection, Construct, Luciferase, Activity Assay, Infection, Control