Increased adherence of flowing monocytes to cultured endothelial cells after hypoxia/reoxygenation: involvement of Rac-1, ROS and VCAM-1

We examined the adhesion of monocytic U937 cells under flow conditions (1 dyne/cm2) to cultured HUVECs subjected to H/R (1h hypoxia/13h reoxygenation). Video microscopy and a parallel-plate flow system were used to measure monocyte-EC adhesive interactions (tethering, rolling, stable adhesion). The number of total interactions was significantly greater (2.5-fold) after H/R, compared to normoxic controls. Either EC pretreatment with the antioxidant pyrrolidine dithiocarbamate (PDTC), a selective NF-kB inhibitor, or adenoviral-mediated gene transfer of a dominant negative Rac-1 gene product (Ad.Rac1N17) significantly decreased the H/R-induced cell rolling and stable adhesion. Since VCAM-1 is a major EC receptor for monocyte adhesion, we examined VCAM-1 expression by whole cell ELISA. While H/R significantly upregulated VCAM-1, both PDTC and Ad.Rac1N17 effectively inhibited VCAM-1 expression.