VEGF165 effect on the fibrinolytic potential of endothelial cells is independent of protein synthesis but depends on uPA activity. (A) When cells were seeded on a fibrin gel (0.5%), VEGF165 (50 ng/mL) stimulation of human umbilical vein endothelial cells (HUVECs) for 60 minutes induces an increase in d-dimer concentrations of cell supernatants, measured by a specific ELISA. The VEGF165 effect was not diminished in the presence of cycloheximide (CHX, 10 μM). Single bars represent mean values of 3 independent experiments; error bars represent SDs. (B) The increase in d-dimer concentrations in supernatants of endothelial cells by VEGF165 (50 ng/mL) is time dependent. Already after 30 minutes a statistically significant higher amount of d-dimer was found in supernatants of VEGF165-stimulated compared with unstimulated endothelial cells (0.292 ± 0.01 mg/L and 0.432 ± 0.04 mg/L; control and VEGF; P < .05). (C) The increase in d-dimer concentration by VEGF165 was partially blocked by an antibody, inhibiting urokinase activity (scuPA14), while an antibody, described to inhibit tPA activity (7VPA), could not diminish the d-dimer concentrations. When urokinase receptor was cleaved by PI-PLC (5 U/mL) or urokinase activity was blocked by the urokinase inhibitor benzamidine (benz, 10 μM), the VEGF effect on d-dimer generation was also diminished. (D) Immunocytochemistry of HUVECs seeded on fibrin gel under serum-deprived conditions, exposed for one hour to VEGF (50 ng/mL) or left untreated as control. After cells were fixed in 4% paraformaldehyde, immunostaining was performed with horseradish peroxidase (POX)-conjugated mouse anti-d-dimer monoclonal antibody followed by streptavidin Alexa-Fluor 488. Samples were mounted in Vectashield. Immunofluorescence microscopy was performed with an Olympus AX70 microscope, whereby digital images were recorded using an F-View camera. Scale bar: 10μm.
