Indeed, during the initial rapid increase in permeability after thrombin stimulation, disruption of adherence junctions between cells, amongst others due to reduced Rac1 activity and subsequently RhoAmediated endothelial contraction , play a role. When the maximum increase in permeability is reached, both disruption of adherence junctions and endothelial contraction play a role. For the current study it was hypothesized that Ang-2 increases basal and thrombin-induced permeability of HPMVECs by impairing vascular endothelial cadherin junctional organization in part via reduced Rac1 and increased RhoA activity. Since Ang-1 has been extensively studied before, Ang-2 data were compared to Ang-1 data. In vitro, endothelial permeability can be evaluated by culturing cells on porous filters and subsequent assessment of the horse radish peroxidase passage or the transendothelial ion-flux via cell-cell and cell-matrix contacts as indicated by the TEER. Since the relationship between the macromolecule flux and the TEER is non-linear and the passage is size-dependent both were assessed. The observation that the angiopoietins modulate in particular the initial response of endothelial cells to thrombin suggests that angiopoietins may affect the molecular organization of the adherence junctions. To study the molecular organization of the adherence junctions, VE-cadherin was visualized by immunofluorescence microscopy as shown at 2 and 15 min after thrombin stimulation in Figures 3a and b, respectively. VEcadherin was encountered in control cells as a continuous and narrow lining at cell-cell borders reflecting Vorinostat HDAC inhibitor stable junctions. Exposure to thrombin induced a redistribution of VE-cadherin into a CPI-613 purchase zigzag wide pattern typical for unstable and activated junctions and the generation of intercellular gaps. Ang-1 preincubation attenuated the effect of thrombin on VE-cadherin redistribution and gap formation. However, still some intercellular gaps were visible in accordance with the thrombin-induced permeability in Ang-1 treated cells. In contrast, the effect of thrombin was enhanced in Ang-2 treated cells, which resulted in even wider VE-cadherin staining and a stronger zigzag pattern together with more intercellular gaps. Ang-1 prevented the enhancement of gap formation in Ang-2 treated cells. The response at 2 and 15 min was similar, although the thrombin-induced gaps were more pronounced at 15 min. The changes in VE-cadherin were accompanied by alterations in the F-actin cytoskeleton. While in control cells most F-actin bundles were seen in the periphery of the cell, thrombin induced the formation of F-actin bundles throughout the cell. However, although there was a counteracting tendency by the presence of Ang-1, the effects of Ang-1 and Ang-2 on thrombin-stimulated stress fiber formation were very limited. We subsequently evaluated tyrosine phosphorylation of VEcadherin , with specific emphasis on that of the tyrosine 685 residue, since this residue is phosporylated by Src , which is linked to angiopoietin signaling in the context of vascular endothelial growth factor -induced endothelial permeability.