Recently it has been found that treatment of

Recently, it has been found that treatment of human phagocytic Ciclopirox ethanolamine mg or microvascular endothelial cells with NO donors inhibited the O2− generating capacity of NADPH oxidase [[8], [9], [10], [11]], which may involve down-regulation or S‑nitrosylation of the p47phox subunit (a cytosolic subunit of NADPH oxidase) [9,10,[12], [13], [14], [15]]. In addition, previous studies have revealed that exposure of phagocytic or endothelial cells to exogenous NO induces expression of heme oxygenase-1 (HO-1) [11,[16], [17], [18], [19]]. HO-1 is involved in catalyzing the oxidative degradation of free heme to liberate ferrous iron, carbon monoxide and biliverdin. The released ferrous iron can catalyze the generation of highly toxic hydroxyl radical from H2O2 via Fenton reaction [17,19]. To prevent this, ferrous iron is incorporated into ferritin and transported out [19], and the released free heme is eliminated by HO-1 activity. In most mammalian cells, biliverdin is converted to bilirubin by biliverdin reductase [19,20]. Both biliverdin and bilirubin, the bile pigments, function as antioxidants to scavenge and detoxify ROS. Therefore, several lines of evidence have indicated that induction of HO-1 has remarkable antioxidant effects and has potent protective actions against ROS-induced oxidative damage both in vitro and in vivo [11,16,[20], [21], [22], [23]]. Moreover, increased expression of HO-1 and subsequent bilirubin production may modulate endogenous cellular ROS generation. It has been demonstrated that HO-1 expression in macrophages inhibited NADPH oxidase activity through decreased heme availability and protein abundance of the heme-containing subunits of NADPH oxidase [24]. It has also been suggested that activation of the HO-1/bilirubin pathway may interact with ROS generating systems in vascular tissues. For example, HO-1 expression reduced NADPH oxidase-dependent ROS production in vascular endothelial cells, and this effect was blocked by the HO-1 inhibitor tin protoporphyrin-IX and mimicked by bilirubin [11]. Induction of HO-1 by hemin (i.p. injection) in mice suppresses NADPH oxidase-derived oxidative stress in the aorta and kidney. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH oxidase-derived O2− production [16]. The vascular endothelium is an active tissue capable of responding to a variety of physiological stresses leading to the modulation of many enzymatic activities, such as the expression of HO-1 [11,16,20,25]. The experiments of the present study were designed to test the hypothesis that NO released from NO donors might modulate NADPH oxidase-derived O2− formation via induction of HO-1 in vascular endothelial cells. We demonstrated that NO significantly attenuated NADPH oxidase-derived O2− generation via a HO-1-dependent mechanism, and we proposed that NO/HO-1 pathway played a critical role in the cytoprotection against hydrogen peroxide-induced endothelial dysfunction.
Material and methods
Results
Discussion In this study, we confirmed that NO could induce HO-1 expression in vascular endothelium and provided a novel mechanism by which NO modulated NADPH oxidase-dependent O2− production. This mechanism for the suppression of NADPH oxidase activity was supported by the following findings: (1) NO donor significantly induced HO-1 expression (Fig. 1A); (2) similar to the effects of NADPH oxidase activity inhibitor, NO donor suppressed O2−-generating capacity of NADPH oxidase (Fig. 1B), an effect distinct from a direct neutralizing action of NO; (3) the inhibitory effect of NO was abolished by the specific small-interfering RNA against HO-1 expression or the co-treatment with HO-1 inhibitor (Fig. 1B and C); and (4) bilirubin, the metabolic product of HO-1, mimicked the effect of NO (Fig. 2B). The current results did not exclude the possibility that NO suppressed NADPH oxidase activation via other mechanisms. Direct scavenging of O2− by the NO was possible [1,7], and NO-mediated S-nitrosylation of critical thiols and down-regulation of subunits in NADPH oxidase was also plausible [9,10,[12], [13], [14], [15]]. Therefore, induction of HO-1 was an alternative mechanism for NO-mediated inhibition of NADPH oxidase function in vascular endothelium.