In addition, Mammoto et al. pointed towards an increased activity of the inhibitory GTPase activating protein p190 RhoGAP as a contributor to the inhibitory effect of Ang-1 on endotoxinmediated vascular leakage. As thrombin induces RhoA activity, a similar mechanism may contribute to the effects observed in the present HPMVECs. Activation of p190RhoGAP by Ang-1 limits the activation of Rho kinase and mDia, which can affect subsequent pathways that enhance permeability. Indeed, Ang-1 caused a reduction in RhoA activation when assayed 15 min after thrombin stimulation, conform Mammoto et al., but not at earlier time points. Therefore, modulation of RhoA activity becomes in particular important when the junctions were already destabilized by the initial response. To our knowledge, we are the first to demonstrate that Ang-2 enhanced AbMole L-Ornithine thrombin-induced endothelial permeability in HPMVECs, similar to the effect of Ang-2 on VEGF-induced retinal endothelial cell permeability. Interestingly, Ang-2 enhanced the initial permeability in particular, suggesting that Ang-2 AbMole Ellipticine modulates the stability of the junctions before or during the initial rapid increase in thrombin-induced permeability, but has less effect during the later phase of the cell contraction after formation of stress fibers, i.e. when the junctional multimeric Ang-1/Tie-2 complexes had disappeared. Indeed, Ang-2 induced a change in the molecular organization of the junctions as demonstrated by an enhancement of the zigzag pattern, while it did not enhance the number or organization of stress fibers during thrombin stimulation. Ang-2 did not enhance VE-cadherin phosphorylation at tyrosine 685, as seen in other conditions. However, the availability of Tyr685 depends on Csk binding, while other VE-cadherin tyrosine residues may be phosphorylated by Ang-2. Alternatively, Ang-2 may act by preventing protective actions on adherence junction proteins. In line with this suggestion, Seegar et al. reported that Ang-2 enhances Tie-1-Tie-2 interaction, which inhibits the endothelial protective effect of Tie-2 activation. This in contrast to Ang-1, which directs protective Tie-2 activity by homomultimerization. This latter action of Ang-1 probably also explains why the combination of equal concentrations of Ang-1 and Ang-2, which in most studies have equal affinities for the Tie2 receptor, still enhanced the initial rate of the thrombin-induced permeability, albeit slightly less than Ang-2 alone. Whether the withdrawal of Tie-2 from junctional multimerization also causes the increase in thrombin-induced hyperpermeability when only Ang-2 is added, is uncertain, because endothelial cells produce little Ang-1 themselves. Signaling by direct interaction of Ang-2 with Tie-1 into the endothelial cell has also been reported and may affect junction stability in thrombinstimulated cells. Finally, Ang-2 can activate endothelial cells via other phosphorylation sites on the Tie2 receptor.