Integrin engagement by vitronectin evokes Ca 2+ elevation and nitric oxide synthesis in human vascular endothelial cells

The stimulation of vascular integrins by ECM‐associated molecules can lead to changes in arterial tone through the generation of local vasoactive signals. In this study, we have examined how endothelial cell integrins may contribute to the regulation of vascular tone via nitric oxide (NO) signaling. Using cultured HUVECs (EA.hy926 cell line) loaded with either 2 μM Fluo‐4 or 1 μM DAF‐FM, we observed that acute exposure of HUVECs to purified human vitronectin (~0.5 mg/ml) produced modest elevations in cytosolic free Ca 2+ (40–50% above baseline) in 10/12 experiments. Conversely, exposure of HUVECs to human fibronectin (~0.5 mg/ml) evoked Ca 2+ increases in only 4/10 experiments. The purinergic agonist UTP (0.5 μM) stimulated robust Ca 2+ elevations in all cells examined. Pre‐treatment of cells with the αVβ3 integrin antibody LM609 blocked vitronectin‐induced Ca 2+ mobilization, whereas similar pre‐treatment with an antibody recognizing the α5β1 integrin (i.e. JBS5) did not interfere with the vitronectin‐stimulated Ca 2+ elevation. Importantly, both vitronectin and UTP were observed to stimulate acute NO production in HUVECs, and the vitronectin‐evoked response was inhibited by pretreatment with the LM609 antibody. Collectively, these data suggest that αVβ3 integrins may be primarily responsible for mediating the observed vitronectin‐evoked increases in cytosolic Ca 2+ and acute NO synthesis.