Nitric oxide donor-induced persistent inhibition of cell adhesion protein expression and NFκB activation in endothelial cells

Nitric oxide (NO), applied by inhalation or released from NO donors, has been used to reduce the expression of cell adhesion molecules (CAM) and ameliorate other consequences of ischemia/reperfusion (I/R) injury. In this study, we have assessed the time frames of pretreatment and of the duration of the preconditioned state using human umbilical vein endothelial cells (HUVECs) and the NO donor, SNAP, in combination with cysteine. The induction of vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM) and E-selectin by the cytokines TNFα and IL-1β, and by bacterial lipopolysaccharide (LPS) was reduced by SNAP/Cys preincubation (30 min, 1 mM) to less than 10% of controls. This refractory state in respect to cytokine-induced CAM expression persisted for 6 h after washout of the NO donor in the combination TNFα/VCAM, and a partial block was still observed after 8 h. The effect was not mediated by the cGMP pathway, as was demonstrated by using the inhibitor of guanylyl cyclase, ODQ, and the cGMP analogue, 8-Br-cGMP. The TNFα-induced expression of CAM was exclusively dependent on the transcription factor NFκB since the inhibitor of NFκB activation, BAY 11-7082, completely blocked the induction. The TNFα-induced phosphorylation and degradation of the inhibitor of κB (IκBα) was suppressed for up to 8 h after SNAP/Cys pretreatment. The inhibitory S-nitrosation of IκB kinase (IKKβ), as assessed by the biotin-switch-procedure and immunoprecipitation, was only detectable immediately after SNAP/Cys incubation but not at later time points. In summary, a short preincubation of HUVEC with SNAP/Cys results in a persistent suppression of NFκB-dependent expression of CAM. The stabilization of IκBα over the same time span may be causally related to this effect.