We demonstrated that bilirubin the product of
We demonstrated that bilirubin, the product of heme metabolism by HO-1, exerted a potent suppression of NADPH oxidase activity (Fig. 2B). Different from the down-regulation of NADPH oxidase subunit by HO-1 induction in macrophages , the protein levels of the NADPH oxidase subunits in vascular endothelium were not altered after HO-1 induction by NO (Fig. 1E), suggesting that, in vascular endothelial cells, modulation of subunit expression was unlikely to be involved in NO-induced inhibition of NADPH oxidase function. Moreover, the translocation of cytosolic p47phox subunit to plasma membrane is essential for the assembly of the active NADPH oxidase and the subsequent burst of O2− production [, , ]. Here, we demonstrated that bilirubin pretreatment seemed to suppress p47phox intracellular redistribution (Fig. 2C), indicating that bilirubin might impact the assembly and activation of endothelial NADPH oxidase. Therefore, our data raise the alternative possibility that the inhibition of NADPH oxidase by HO-1 may be mediated by its product bilirubin, consistent with previous studies that exogenous bilirubin inhibited NADPH oxidase-dependent O2− production in neutrophil cells, smooth muscle cells and isolated JNJ 5207852 dihydrochloride . These actions of bilirubin were, however, independent of ROS scavenging, because the O2− scavenging effect of bilirubin was weak [11,16] and the inhibition of NADPH oxidase activity by bilirubin persisted after the cells had been washed (Fig. 2B). Although our results suggested that bilirubin had a pivotal role in the antioxidant effects of HO-1, other intermediate products of HO-1, such as carbon monoxide or biliverdin [16,29], might also be involved in the suppression of NADPH oxidase activity. Interestingly, there is evidence of an inverse relationship between the serum bilirubin level and risk of cardiovascular disease, indicating that bilirubin may act as an endogenous cardiovascular protective factor [16,30,31]. We propose that suppression of NADPH oxidase activation may underlie this cardiovascular protection. Here we also observed that NO suppressed oxidative stress-induced endothelial dysfunction through HO-1 induction. Vascular endothelial dysfunction is the earliest event in the pathogenesis of cardiovascular diseases [, , , ]. Given that both nitric oxide synthase and HO-1 are upregulated in vascular inflammation, such as atherosclerosis [11,20], it has been suggested that HO-1 induction by NO could be a physiological compensatory mechanism that may protect endothelial cells from exaggerated oxidative injury during inflammation by modulating NADPH oxidase activity. During inflammation or oxidative stress, excess H2O2 would induce significant endothelial dysfunction such as loss of cell viability, increase of ROS and eNOS uncoupling, and NO donors conferred protection against oxidative stress to vascular endothelium (Fig. 3). In addition, the protective effects of NO was associated with NO-mediated activation of HO-1 and a reduction of NADPH oxidase activity (Fig. 3B). In agreement with these in vitro data, aortic NADPH oxidase activity was increased in NAME-treated mice while the endothelial function was simultaneously impaired (Fig. 4), further revealing the possible inhibition of endogenous NO on vascular oxidative stress and endothelial dysfunction in vivo. It should be noted that NO deficiency in vivo did not reduce HO-1 expression, although inhibition of NO formation by NAME treatment increased NADPH oxidase activity. This is not surprising, because the increased oxidative stress in vascular tissues can stimulate the expression of HO-1, a critical stress response enzyme [20,25]. This intracellular adaptation represents the ability of endothelial cells to acclimate themselves to new conditions to maintain vascular homeostasis. In summary, it was shown herein that NO might modulate NADPH oxidase-dependent O2− production in vascular endothelium, at least partly by inducing HO-1. Moreover, NO/HO-1 pathway played a critical role in the cytoprotection against oxidative stress-induced endothelial dysfunction. This inhibition of NADPH oxidase activity by HO-1 induction may represent a novel mechanistic link between the cardiovascular protective actions of NO and suppression of oxidative stress. Inducing the NO/HO-1 pathway to target NADPH oxidase might be a new therapeutic approach for prevention and treatment of vascular diseases.