TI - Bleo and SS increase oxidative stress and induce iNOS -dependent NO synthesis . AB - As part of its physiological mechanism of action , laminar flow increases the intracellular content of NO by eNOS activation[22] . In our experiments SS increased about 2.5 fold , above basal level , the production of NO in cells kept in normal condition , however , this effect was further enhanced up to 4.5 fold by SS administered in the presence of Bleo ( Fig 2a ) . In patho-physiological contexts , oxidative stress or hypoxia increase the expression of the inducible NOS isoform which synthesizes high levels of NO[23] independently of PHOSphorylation signals . Figure 2b , left and right panels , shows that iNOS expression was induced about 2.5.fold above control by Bleo and even further by the coincident treatment of SS and Bleo . We reasoned that these results could be explained by the Bleo -dependent production of ROS which could be intensified in the presence of SS . Supplemental Figure S1 , panels a and b , shows that Bleo and SS in combination determined a significant increase in the intracellular content of oxygen free radicals evaluated by dichlorofluorescein ( DCF ) and dihydroethidium ( DHE ) staining . Notably , DCF is also able to detect NO[24] which may explain the significant increase in fluorescent signals occurring in the presence of SS and detectable in absence of Bleo as depicted in the supplemental Figure S1 , panel a . To evaluate the contribution of NO to the SS -dependent endothelial toxicity occurring in the presence of Bleo an experiment was performed in HUVEC kept in static culture and treated with Bleo in the presence of the NO donor DETA/NO . Figure 2c , shows that the NO donor significantly increased cell death indicating that , even in the absence of flow , NO delivered to cells with an elevated intracellular content of ROS contributed to the onset of endothelial toxicity . To investigate the role of iNOS in this process a series of experiments were performed in which the iNOS specific inhibitor GW274150 was added to the culture medium in the presence or absence of Bleo with or without SS . Figure 2d shows that GW274150 inhibited the pro-apoptotic response triggered by laminar flow and Bleo . Further , the iNOS inhibitor , virtually abolished the SS -dependent production of NO ( Figure 2e ) suggesting that , in this experimental setting , iNOS may be accounted as the major source of NO . Although NO is an important positive regulator of endothelial cell function its dual role in apoptosis and survival is well known[11] . In fact , while NO itself is fairly non-toxic and protective , the secondary reactive nitrogen species generated in the presence of free oxygen radicals are potent oxidant and nitrating agents that can modify the structure and function of numerous biomolecules both in vitro , and in vivo[25] , [10] . Specifically , in the presence of ROS , the newly synthesized NO easily reacts with them forming highly damaging RNS including peroxynitrites[11] . Figure 2f , left and right panels , shows that in the presence of Bleo and laminar SS the total intracellular content of proteins bearing nitrotyrosine residues increased about two fold compared to controls as indicated by western blotting and densitometric analyses . One of the most common consequences of peroxynitrites formation and protein nitration is the functional alteration of important signalling transducers such as the inactivation of PI3K and that of other pro-survival molecules[11] , [25] followed by the activation of pro-apoptotic mechanisms which may be relevant to the endothelial cell death process described in this manuscript[11] .