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The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration.

Heme oxygenase, the rate-limiting step in heme degradation to CO and bilirubin, exists in inducible (HO-1) 1 and constitutive (HO-2 and HO-3) isoforms, the synthesis and activities of which are differentially regulated in mammalian tissues (1-3). The common conception that these enzymes are merely components of a catabolic pathway that facilitates the elimination of toxic products from the organism has been disputed by strong evidence demonstrating that endogenously generated CO and bilirubin act as crucial effector molecules in the mitigation of vascular and cellular dysfunction (4 -12). Disparate conditions and a number of pathological states including hypoxia, endotoxic shock, atherosclerosis, and inflammation have been found to promote overexpression of the HO-1 gene and increased heme oxygenase activity (13-18). Although the molecular mechanism(s) leading to HO-1 induction by these and other conditions remains to be fully elucidated, a common denominator that characterizes the prompt stimulation of HO-1 under most circumstances is the transient decrease in cellular glutathione levels and a drastic change in the redox status of the intracellular milieu (15, 19 -21). It is not surprising, therefore, that conditions associated with increased production of reactive oxygen species and reactive nitrogen species (RNS) favor the activation of the HO-1/CO/bilirubin pathway, which is now regarded as an important cellular stratagem to counteract and resist different stress insults (3,22). In the context of redox reactions and signal transduction events that elicit the expression of HO-1 in vascular tissue, the gaseous molecule NO has recently been highlighted as an important biological modulator (see reviews in Refs. 22 and 23; Refs. 15 and 24). NO has been implicated in a wide range of processes critical to normal functions in the cardiovascular, nervous, and immune systems; the cytotoxic nature of NO has also been extensively emphasized when excessive production of this gas is triggered under certain pathological conditions (25). The conception that HO-1 might function to counteract the potential toxic effects evoked by NO first emerged from the discovery that certain NO-releasing agents can stimulate an increase in HO-1 transcript and heme oxygenase activity, resulting in protection against oxidative stress (26 -28). Subsequent reports have confirmed these findings (24, 29 -31), and more recent works have established that NO-related species (such as peroxynitrite and S-nitrosoglutathione) as well as endogenously generated NO and S-nitrosothiols are also capable of 32). In light of the rather complex and diverse chemistry of the NO group, which enables it to exist in a variety of interrelated redox-activated forms, investigations are now required to explore which additional NO * This work was supported by British Heart Foundation Grants PG/ 1999-005 and PG/2001-037 (to R. M.) and PG/2000 and by funds from the Dunhill Medical Trust. The National Heart Research Fund and the Wellcome Trust provided travel grants to p...

NO potently up-regulates vascular haem oxygenase-1 (HO-1), an inducible defensive protein that degrades haem to CO, iron and the antioxidant bilirubin. Since several pathological states are characterized by increased NO production and liberation of haem from haem-containing proteins, we examined how NO influences HO-1 induction mediated by haemin. Aortic endothelial cells treated with S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP) or diethylenetriamine-NONOate (DETA/NO) and haemin exhibited higher levels of haem oxygenase activity compared with cells exposed to NO donors or haemin alone. This was accompanied by a marked increase in bilirubin production and, notably, by a strong magnification of cellular haem uptake. A role for haem metabolites in modulating HO-1 expression by NO was assessed by exposing cells to SNAP, SNP or DETA/NO in medium derived from cells treated with haemin, which contained increased bilirubin levels. This treatment considerably potentiated HO-1 expression and haem oxygenase activity mediated by NO and the use of a haem oxygenase inhibitor abolished this effect. Both iron liberated during haem breakdown and the formation of nitroxyl anion from NO appeared to partially contribute to the amplifying phenomenon; in addition, medium from haemin-treated cells significantly augmented the release of NO by NO donors. Thus we have identified novel mechanisms related to the induction of HO-1 by NO indicating that the signalling actions of NO vary significantly in the presence of haem and haem metabolites, ultimately increasing the defensive abilities of the endothelium to counteract oxidative and nitrosative stress.

Mitochondria-directed antioxidants 2-5 were designed by conjugating curcumin congeners with different polyamine motifs as vehicle tools. The conjugates emerged as efficient antioxidants in mitochondria and fibroblasts and also exerted a protecting role through heme oxygenase-1 activation. Notably, the insertion of a polyamine function into the curcumin-like moiety allowed an efficient intracellular uptake and mitochondria targeting. It also resulted in a significant decrease in the cytotoxicity effects. 2-5 are therefore promising molecules for neuroprotectant lead discovery.

Objective-Sickle cell disease (SCD) is characterized by extensive hemolysis, increased cellular adhesion, and vaso-occlusion. Tissues from sickle patients express heme oxygenase-1 (HO-1), the enzyme that degrades free heme/hemoglobin to the signaling molecule carbon monoxide, and the antioxidants biliverdin/bilirubin. Here, we examined the HO response in endothelial cells exposed to human sickle blood and determined whether this response is beneficial for SCD. Methods and Results-We measured HO activity in human and bovine aortic endothelial cells incubated with human sickle or normal blood. Sickle blood increased HO activity, which was enhanced by hypoxia and was caused mainly by the red cell components of sickle blood. Oxidized hemoglobin was higher in sickle blood and increased markedly over time. Interestingly, HO activity correlated inversely with patients' hemoglobin levels and positively with bilirubin and lactate dehydrogenase. HO-1 induction, exogenous biliverdin, or carbon monoxide markedly decreased adhesion of sickle blood to the endothelium, and sickle red cells partially inhibited relaxation mediated by carbon monoxide in isolated aortas. Key Words: adhesion Ⅲ carbon monoxide Ⅲ endothelium Ⅲ heme oxygenase Ⅲ sickle cell disease S ickle cell disease (SCD) is caused by a mutation in the ␤-globin gene, resulting in abnormal hemoglobin (HbS), either in a homozygous or in a compound heterozygous form with another atypical hemoglobin. The mutated HbS polymerizes in the deoxygenated state, leading to unusually shaped red cells. Sickle erythrocytes hemolyze easily, causing chronic anemia and small vessel occlusion, triggering acute painful episodes and other complications of SCD, such as acute chest syndrome, avascular necrosis, leg ulcers, and renal failure. 1,2 Presently, hydroxyurea is the only drug widely used for treating SCD. Hydroxyurea stimulates production of fetal hemoglobin and lowers the concentration of HbS; 3 however, most patients receiving it still exhibit hemolysis and an underlying inflammatory state, 4 indicating the need for new therapeutic strategies to help patients with SCD. Conclusions-OurDespite a well-characterized genetic origin, SCD may be viewed as a chronic inflammatory condition, 4 -6 with studies revealing abnormally high levels of leukocytes in subjects with SCD. 7 Circulating endothelial cells from SCD patients also display an activated state with expression of adhesion molecules, 8 and a genetic profile of blood mononuclear cells from 27 patients in steady-state SCD showed upregulation of genes related to oxidative stress and inflammation and global upregulation of proinflammatory markers. 4 Interestingly, genes of heme metabolism, including heme oxygenase-1 (HO-1) and biliverdin reductase, were also increased, as already reported for circulating endothelial cells and kidneys of SCD subjects. 9 HO-1 is the inducible isoform of HO, the enzyme that converts heme to iron, carbon monoxide (CO), and biliverdin. Biliverdin is subsequently reduced to bilirubin by biliverdin redu...

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO ϩ and NO Ϫ donors interacted with reduced (HbA 0 ) or oxidized (metHb)