Abstract 425: Intermedin Induces Restoration of Endothelial Barrier Function via Rac1-Mediated Rearrangement of Actin Cytoskeleton and VE-Cadherin
Intermedin (IMD), a novel member of calcitonin gene-related peptide superfamily, acts via calcitonin receptor like receptors (CRLR) coupled to adenylyl cyclase. It has been reported that IMD protects against inflammatory mediators-induced endothelial barrier failure via cAMP/PKA signalling pathway. Inflammatory mediators like thrombin induce failure of endothelial barrier function, which is followed by slow recovery, however, the precise mechanism is unknown. Here, the molecular mechanism regulating IMD-mediated restoration of endothelial barrier function was analysed.
Methods: In cultured human umbilical vein endothelial cells (HUVEC), the effects of thrombin (0.2 U/ml) and IMD (10 nM) on monolayer permeability (albumin flux), RhoA, Rac1 and cdc42 activation (pulldown assay), actin cytoskeleton and VE-cadherin (confocal microscopy) translocation and contractile activation (myosin light chain phosphorylation) were analyzed
Results: Exposure of HUVEC to thrombin lead to a rapid increase in endothelial macromolecule permeability (2 ± 0.5-Fold) (n = 5; P < 0.05, for all further parameters), activation of RhoA (3 ± 0.5-Fold), inactivation of Rac1, however, cdc42 was not affected. It also induced stress fiber formation and contractile activation, reduced cortical actin and delocalized VE-cadherin from cell-cell contacts. Loss of barrier function was followed by slow recovery of barrier function over 90 minutes accompanied by RhoA and contractile inactivation, Rac1 activation, and appearance of cortical actin and VE-cadherin. Addition of IMD induced a fast recovery of barrier function to basal level within 10 minutes accompanied by RhoA (52 ± 9%) and contractile inactivation (72 ± 9%), Rac1 activation (3 ± 0.5-Fold) (however, cdc42 was unaffected) and appearance of cortical actin and VE-cadherin at cell-cell contacts. These IMD effects were abolished by CGRP19–37 (1 μM, CRLR antagonist). Inhibition of Rac1 by NSC23766 (100 μM) and LT82 (20 ng/ml), Rac1 inhibitors, abolished IMD-mediated recovery of cytoskeleton, VE-cadherin and barrier function.
Conclusion: These data demonstrate that IMD restores thrombin-induced barrier failure through rearrangement of cortical actin cytoskeleton and VE-cadherin via Rac1 activation.