Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells.

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.

Human sickle cell blood modulates endothelial heme oxygenase activity: effects on vascular adhesion and reactivity.

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. CONCLUSIONS: Our results highlight important associations between SCD and HO byproducts, which may counteract vascular complications of SCD.

CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule.

Carbon monoxide (CO) is emerging as an important and versatile mediator of physiological processes to the extent that treatment of animals with exogenous CO gas has beneficial effects in a range of vascular- and inflammatory-related disease models. The recent discovery that certain transition metal carbonyls function as CO-releasing molecules (CO-RMs) in biological systems highlighted the potential of exploiting this and similar classes of compounds as a stratagem to deliver CO for therapeutic purposes. Here we describe the biochemical features and pharmacological actions of a newly identified water-soluble CO releaser (CORM-A1) that, unlike the first prototypic molecule recently described (CORM-3), does not contain a transition metal and liberates CO at a much slower rate under physiological conditions. Using a myoglobin assay and an amperometric CO electrode, we demonstrated that the release of CO from CORM-A1 is both pH- and temperature-dependent with a half-life of approximately 21 min at 37 degrees C and pH 7.4. In isolated aortic rings, CORM-A1 promoted a gradual but profound concentration-dependent vasorelaxation over time, which was highly amplified by YC-1 (1 microM) and attenuated by ODQ, a stimulator and inhibitor of guanylate cyclase, respectively. Similarly, administration of CORM-A1 (30 micromol/kg i.v.) in vivo produced a mild decrease in mean arterial pressure, which was markedly potentiated by pretreatment with YC-1 (1.2 micromol/kg i.v.). Interestingly, an inactive form of CORM-A1 that is incapable of releasing CO failed to promote both vasorelaxation and hypotension, thus directly implicating CO as the mediator of the observed pharmacological effects. Our results reveal that the bioactivities exerted by CORM-A1 reflect its intrinsic biochemical behavior of a slow CO releaser, which may be advantageous in the treatment of chronic conditions that require CO to be delivered in a carefully controlled manner.

Polyamine conjugation of curcumin analogues toward the discovery of mitochondria-directed neuroprotective agents.

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.

The interaction of nitric oxide with distinct hemoglobins differentially amplifies endothelial heme uptake and heme oxygenase-1 expression.

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+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.

Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion.

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.

Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway.

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.