1Justus-Liebig-Universität Giessen Experimentelle Kardiologie Gießen, Deutschland
Background: Dorsomorphin is a well-known pharmacological inhibitor of bone morphogenic (BMP) and vascular endothelial growth factor (VEGF) signalling. We and others have previously demonstrated dorsomorphin as an antiangiogenic factor in endothelial cells. In the present study, we investigated the effects of dorsomorphin on intracellular calcium and its role in modulation of endothelial adherens junctions and barrier function.
Methods: The study was carried out on cultured human umbilical vein endothelial cells (HUVEC). HUVEC permeability was analysed by measuring flux of albumin across HUVEC monolayers cultured on filter membranes. Intracellular calcium (Ca2+) was measured by Fura-2 ratio using fluorescence live-cell imaging. Endothelial adherens junctions were visualised by immunocytochemistry followed by fluorescence microscopy. Rac1-activation was analysed by GTPase pulldown assay.
Results: Dorsomorphin reduced basal endothelial permeability in a concentration-dependent manner achieving maximum effect with 10 mM and antagonised thrombin-induced endothelial hyperpermeability. The endothelial barrier-stabilising effect was accompanied by robust activation of Rac1 GTPase and recruitment of actin and VE-cadherin at cell-cell (adherens) junctions. Over-expression of either dominant negative or pharmacological inhibition of Rac1 abrogated dorsomorphin effect on endothelial-adherens junctions and barrier function. Moreover, dorsomorphin induced an increase in cytosolic Ca2+-levels in a concentration-dependent manner which was not blocked by removing extracellular Ca2+ concentration suggesting release of Ca2+ from internal stores. Sequestering intracellular Ca2+ using a calcium chelator BAPTA-AM but not removing extracellular calcium abrogated dorsomorphin-induced Rac1 activation and stabilisation of endothelial adherens junctions and barrier function. Finally, investigating the mechanism of dorsomorphin-mediated Rac1-activation, it was observed that dorsomorphin-induced Ca2+-release mediated an activation of Rap1-GTPase upstream of Rac1.
Conclusion: In the present study, we first time demonstrate the effect of dorsomorphin on endothelial Ca2+ signalling and its link to dorsomorphin-induced Rac1 activation and stabilisation of endothelial adherens junctions.