| Abstract: | A permissive role of nitric oxide (NO) in endothelial cellmigration and angiogenesis promoted by vascular endothelial growth factor (VEGF), endothelin, and substance P has previously been established. The present studies were designed to examine the mechanism(s) involved in the NO effect on focal adhesions. Time-lapse videomicroscopy of human umbilical vein endothelial cells (HUVECs) plated on the silicone rubber substrate revealed that unstimulated cells were constantly remodeling the wrinkling pattern, indicative ofchanging tractional forces. Application of NO donors reversibly decreased the degree of wrinkling, consistent with the release oftractional forces exerted by focal adhesions and stress fibers. Morphometric and immunocytochemical analyses showed that NO inhibited adhesion and spreading of HUVECs and attenuated recruitment of paxillinto focal adhesions. NO also had a profound dose-dependent effect on theformation of stress fibers by HUVECs. De novo formation of focaladhesions in HUVECs was significantly diminished in the presence of NOdonors. Migration of HUVECs showed an absolute requirement for thefunctional NO synthase. NO donors did not interfere with focal adhesionkinase recruitment to focal adhesions but affected the state of itstyrosine phosphorylation, as judged from the results ofimmunoprecipitation and immunoblotting experiments. Videomicroscopy ofHUVECs presented with VEGF in a micropipette showed that the rate ofcell migration was slowed down by NO synthase inhibitionas well as by inhibition of tyrosine phosphorylation. Collectively,these data indicate that NO reversibly releases tractional forcesexerted by spreading endothelial cells via interference with the denovo formation of focal adhesions, tyrosine phosphorylation ofcomponents of focal adhesion complexes, and assembly of stress fibers. |
