Abstract 1026: Intermedin induces failure of Microvascular Endothelial Barrier via derrangement of Actin Cytoskeleton and dephosphorylation of Focal Adhesion Proteins
Intermedin (IMD) is a novel member of calcitonin gene-related peptide family which has been shown to be released by endothelial cells (EC) and acts via increased production of cAMP via calcitonin receptor like receptor (CRLR) family coupled to adenylyl cyclase. Recently it has been shown by us that maneuvers increasing intracellular levels of cAMP cause stabilisation of endothelial barrier in macrovascular aortic EC but in contrast it induces failure of barrier function in microvascular coronay EC. Therefore, here the effect of IMD on endothelial barrier function of coronary microvascular EC was studied.
Methods and Results: In cultured coronary microvascular EC monolayers IMD (10 nM) increased permeability (albumin flux) by 145 ± 7% and reduced phosphorylation of myosin light chains (a key regulator of EC contractile apparatus) by 65 ± 6% (western blot; n = 5, P<0.05, for all further parameters). IMD caused inactivation of small GTPases, RhoA and Rac1 (pulldown assay), the key regulators of EC cytoskeleton, by 0.5-fold and 0.6-fold respectively. This inhibition was accompanied by dissassembly of actin filaments (confocal microscopy), dephosphorylation of paxillin and FAK (focal adhesion proteins) by 52 ± 6% and 40 ± 5%, respectively (western blot) and loss of VE-cadherin from cell-cell adhesions leading to rapid stellation of EC. These IMD effects were blocked by a CRLC inhibitory peptide (CGRP8 –37; 100 nM) and a PKA inhibitor (H89; 10 μM). Accordingly, forskolin, a direct activator of adenylyl cyclase, mimicked these IMD effects. Inhibition of protein tyrosine phosphatases (PTPs) with orthovanadate (500 μM) abolished IMD-induced EC barrier failure, Rac1 inactivation as well as paxillin and FAK dephosphorylation, but had no effect on RhoA inactivation. Inhibition of PTPs lead to activation of Rac1 and the rearrangement of actin cytoskeleton at cell periphery and translocation of VE-cadherin at cell-cell adhesions.
Conclusion: The data of present study demonstrate that in microvasculature EC, IMD induces barrier failure, mainly by inactivation of Rac1, RhoA, and dephosphorylation of paxillin and FAK. This leads to dissassembly of actin filaments, loss of cell-cell adhesions, and inactivation of focal adhesions proteins.