Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the beta1- and beta2-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1beta-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation.

Objective-Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. Methods and Results-The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. Conclusion-PMCs