| - BACKGROUND: The onset of atherosclerosis is dominated by inflammatory events which include the activation of the endothelium and increased adhesion and extravasation of activated leukocytes. The adhesion and transendothelial migration is crucially dependent on interactions between leukocyte integrins and immunoglobulin-like cell adhesion molecules (Ig-CAMs), such as ICAM-1 and VCAM-1, on the endothelium. VCAM-1 is of particular interest as it was recently shown to play a major role in the early stages of atherosclerosis. We have recently identified new VCAM-1-mediated signaling which controls endothelial integrity and is required for monocyte transendothelial migration. This signaling pathway comprises activation of the small GTPase Rac, production of reactive oxygen species and activation of p38 MAP kinase and leads to a loss of VE-cadherin-mediated cell-cell adhesion and reduced endothelial integrity. It is unknown how VCAM-1 engagement triggers this signaling pathway but recent studies suggest that Ig-CAMs, including VCAM, can associate with other transmembrane molecules on the same cell, including receptor tyrosine kinases and cadherins, thus forming multi-molecular signaling complexes. - HYPOTHESIS: VCAM-1 triggers signaling to control endothelial integrity and leukocyte transmigration by associating with (transmembrane) receptors that harbour intrinsic enzymatic activity and provide docking sites for signaling proteins. We will define the signaling at the plasma membrane, initiated upon antibody-mediated crosslinking of VCAM-1(as a mimic of monocyte adhesion), using biochemical and immunological assays and we will identify the molecules involved. These studies will focus on a role for ERM (Ezrin, Radixin, Moesin) proteins, receptor tyrosine kinases such as the FGF or VEGF receptor, the FcgammaRIIA and for N-cadherin in their association with VCAM-1 and in the transmission of intracellular signals. The methods will include co-immunoprecipitation and kinase assays, confocal microscopy on fixed and living cells, delineation of endothelial signaling using cell-penetrating proteins and peptides and analysis of endothelial integrity using real-time electrical resistance measurements. The relevance of the identified signaling events will be further established using pharmacological inhibitors or cell-permeable, signaling-blocking peptides in combination with assays for monocyte transendothelial migration. These studies are anticipated to: (i) define the composition of VCAM-associated signaling complexes; (ii) assess the differences in composition and function of such complexes associated with other relevant Ig-CAMs, such as ICAM-1; (iii) assess the role of the induced signaling in VCAM-mediated modulation of endothelial integrity and leukocyte transmigration. - OBJECTIVES: The primary objective of this study is to identify and characterize VCAM-1-associated multi-molecular complexes that are involved in leukocyte-EC interactions and EC activation. The composition and dynamics of these complexes will be identified using biochemical and live-cell fluorescence imaging techniques. The second objective is to identify proximal signaling events, initiated by activation of these complexes. Finally, the relevance of these signaling complexes for endothelial integrity and monocyte transendothelial migration will be established. - RELEVANCE FOR CARDIOVASCULAR DISEASES: The subject of this proposal is directly related to changes in human EC upon association with activated leukocytes. This is a process that is relevant for inflammatory disorders and thus for cardiovascular diseases, as these have been shown to have a clear inflammatory component, especially at the onset of disease. The signaling complexes that will be studied in this project control a crucial feature of endothelial cells: their cadherin-mediated cell-cell adhesion, which is directly related to endothelial integrity and to the rate of leukocyte flux into the tissues. Given the fact that endothelial integrity and function is disturbed in cardiovascular disease, understanding its regulation is important to define possible ways to improve endothelial cell function. The current proposal will use agents that interfere with inflammatory processes mediated by EC. The proposed studies may therefore contribute to the identification and/or development of new therapeutic agents that reduce EC dysfunction and be especially useful in the onset of atherogenesis. |
