Our previous studies using a porcine model have shown that inhalation of NO for a brief period just before the onset of ischemia can protect the lung against ischemia/reperfusion (I/R) injury later on. The protection persisted throughout 90 min ischemia of the left lung and a reperfusion phase of up to 5 h. Here we present first results of the attempt to investigate the underlying mechanisms of “NO preconditioning” by an in vitro approach using endothelial cells. Since NO pretreatment was especially effective in blocking I/R-induced inflammation, the study aims at the cytokine-dependent and NFkappaB-mediated expression of cell adhesion molecules (CAM).
Human umbilical vein endothelial cells (HUVEC) were exposed to the NO donor, SNAP, for 5 min to 60 min. After washout, cells were cultured in reagent-free medium for up to 16 h before the CAM - ICAM, VCAM, and E-selectin - were induced by exposure to TNFalpha or IL-1beta. The CAM on the cell surface were quantified by cell ELISA, and the level of the inhibitor of NFkappaB activation, IkappaBalpha, by Western blotting. The relevance of the cGMP pathway was tested by using the guanylyl cyclase inhibitor, ODQ, and the cGMP analogue, 8-Br-cGMP. The dependence on NFkappaB was verified by the inhibitor of NFkappaB activation, BAY 11-7082.
Preincubation with SNAP (1 mM) for 30 min was sufficient to reduce the cytokine-induced expression of CAM to less than 10 % of controls. This “refractory” state persisted for 6 h after washout of the NO donor in the combination TNFalpha/VCAM. The SNAP effect was not mediated by the cGMP pathway. The TNFalpha-dependent induction of CAM was strictly dependent on the activation of NFkappaB. After TNFalpha exposure, a high level of IkappaBalpha was persistently detected in SNAP-treated HUVEC, while the inhibitor was immediately degraded in controls.
Persistent blocking of NFkappaB-dependent CAM expression by SNAP pretreatment in endothelial cells may be causally related to the stabilization of IkappaBalpha.
Results may be relevant to clinical situations of I/R-induced inflammation.
Thomas Waldow, None.