Colin J. Green

Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element

The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2-Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2-Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway.

Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress

Curcumin, a widely used spice and coloring agent in food, has been shown to possess potent antioxidant, antitumor promoting and anti-inflammatory properties in vitro and in vivo. The mechanism(s) of such pleiotropic action by this yellow pigment is unknown; whether induction of distinct antioxidant genes contributes to the beneficial activities mediated by curcumin remains to be investigated. In the present study we examined the effect of curcumin on endothelial heme oxygenase-1 (HO-1 or HSP32), an inducible stress protein that degrades heme to the vasoactive molecule carbon monoxide and the antioxidant biliverdin. Exposure of bovine aortic endothelial cells to curcumin (5-15 microM) resulted in both a concentration- and time-dependent increase in HO-1 mRNA, protein expression and heme oxygenase activity. Hypoxia (18 h) also caused a significant (P < 0.05) increase in heme oxygenase activity which was markedly potentiated by the presence of low concentrations of curcumin (5 microM). Interestingly, prolonged incubation (18 h) with curcumin in normoxic or hypoxic conditions resulted in enhanced cellular resistance to oxidative damage; this cytoprotective effect was considerably attenuated by tin protoporphyrin IX, an inhibitor of heme oxygenase activity. In contrast, exposure of cells to curcumin for a period of time insufficient to up-regulate HO-1 (1.5 h) did not prevent oxidant-mediated injury. These data indicate that curcumin is a potent inducer of HO-1 in vascular endothelial cells and that increased heme oxygenase activity is an important component in curcumin-mediated cytoprotection against oxidative stress.

Cardioprotective Actions by a Water-Soluble Carbon Monoxide–Releasing Molecule

&NA; Carbon monoxide, which is generated in mammals during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator. Transition metal carbonyls have been recently shown to function as carbon monoxide‐releasing molecules (CO‐RMs) and to elicit distinct pharmacological activities in biological systems. In the present study, we report that a water‐soluble form of CO‐RM promotes cardioprotection in vitro and in vivo. Specifically, we found that tricarbonylchloro(glycinato)ruthenium(II) (CORM‐3) is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution. Cardiac cells pretreated with CORM‐3 (10 to 50 &mgr;mol/L) become more resistant to the damage caused by hypoxia‐reoxygenation and oxidative stress. In addition, isolated hearts reperfused in the presence of CORM‐3 (10 &mgr;mol/L) after an ischemic event displayed a significant recovery in myocardial performance and a marked and significant reduction in cardiac muscle damage and infarct size. The cardioprotective effects mediated by CORM‐3 in cardiac cells and isolated hearts were totally abolished by 5‐hydroxydecanoic acid, an inhibitor of mitochondrial ATP‐dependent potassium channels. Predictably, cardioprotection is lost when CORM‐3 is replaced by an inactive form (iCORM‐3) that is incapable of liberating CO. Using a model of cardiac allograft rejection in mice, we also found that treatment of recipients with CORM‐3 but not iCORM‐3 considerably prolonged the survival rate of transplanted hearts. These data corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO and highlight the bioactivity and potential therapeutic features of CO‐RMs in the mitigation of cardiac dysfunction. The full text of this article is available online at http://www.circresaha.org. (Circ Res. 2003;93:e2‐e8.)

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages

1 The enzyme heme oxygenase‐1 (HO‐1) is a cytoprotective and anti‐inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti‐inflammatory properties of HO‐1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO‐releasing molecules (CO‐RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti‐inflammatory characteristics of CORM‐2 and CORM‐3 in an in vitro model of lipopolysaccharide (LPS)‐stimulated murine macrophages. 2 Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM‐2 or CORM‐3 (10–100 μM) reduced nitrite generation in a concentration‐dependent manner but did not affect the protein levels of iNOS. CORM‐3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. 3 CORM‐2 or CORM‐3 did not cause any evident cytotoxicity and produced an increase in HO‐1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N‐acetylcysteine. 4 CORM‐3 also considerably reduced the levels of tumor necrosis factor‐α, another mediator of the inflammatory response. 5 The inhibitory effects of CORM‐2 and CORM‐3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. 6 These results indicate that CO liberated by CORM‐2 and CORM‐3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.

Thiol Compounds Interact with Nitric Oxide in Regulating Heme Oxygenase-1 Induction in Endothelial Cells INVOLVEMENT OF SUPEROXIDE AND PEROXYNITRITE ANIONS

Thiols are very important antioxidants that protect cells against oxidative insults. Recently, a different and new physiological role has been defined for these compounds because of their involvement in nitric oxide (NO) binding and transport in biological systems. In view of these characteristics, we examined the effect of thiols and NO on the expression of the inducible form of heme oxygenase (HO-1), a stress protein that degrades heme to carbon monoxide and biliverdin. Cultured bovine aortic endothelial cells exposed to the NO donors sodium nitroprusside (SNP) andS-nitroso-N-acetylpenicillamine (SNAP) resulted in increased heme oxygenase activity and HO-1 expression. Co-incubation with N-acetylcysteine, a precursor of glutathione synthesis, significantly attenuated heme oxygenase induction by SNP and SNAP, and a reduction in heme oxygenase activity was also observed when cells were preincubated with N-acetylcysteine for 16 h prior to exposure to NO donors. This effect appears to be associated with NO stabilization by thiols through the formation ofS-nitrosothiols. Hydroxocobalamin, a specific NO scavenger, significantly decreased endothelial heme oxygenase activity, indicating a direct involvement of NO released by NO donors to regulate the expression of this stress protein. Moreover, superoxide anion (O·̄2) and its reaction product with NO, peroxynitrite (ONOO−), were found to partially contribute to the observed NO-mediated activation of endothelial heme oxygenase. Thus, we suggest the existence of a dynamic equilibrium among free NO, O·̄2, and endogenous glutathione, which might constitute an interactive signaling mechanism modulating stress and adaptive responses in tissues.

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 ∼21 min at 37°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 µM) and attenuated by ODQ, a stimulator and inhibitor of guanylate cyclase, respectively. Similarly, administration of CORM‐A1 (30 µmol/kg i.v.) in vivo produced a mild decrease in mean arterial pressure, which was markedly potentiated by pretreatment with YC‐1 (1.2 µmol/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.

Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1

1 The contribution of haeme oxygenase‐derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO‐1). 2 Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl penicillamine (SNAP, 500 μm) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration‐response curve to phenylephrine (PE, 1–3000 nm). 3 Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO‐1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO‐1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells. 4 Induction of HO‐1 in SNAP‐treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2‐14C]‐L‐glycine, the precursor of haeme. Guanosine 3′,5′‐monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO‐1. 5 Incubation of aortic rings with the NO synthase inhibitor, NG‐monomethyl‐L‐arginine (100 μm), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE‐mediated contractility in SNAP‐treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP‐IX, 10 μm), restored the pressor response to PE in SNAP‐treated rings whilst markedly reducing CO and cyclic GMP production. 6 We conclude that up‐regulation of the HO‐1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule

Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO‐releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM‐3) in vitro and in vivo. CORM‐3 produced a concentration‐dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25–50 μM. Inactive CORM‐3, which does not release CO, did not affect vascular tone. Blockers of ATP‐dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM‐3‐dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM‐3 stimulation. The in vitro and in vivo vasorelaxant activities of CORM‐3 were further enhanced in the presence of YC‐1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM‐3, suggesting that factors produced by the endothelium influence CORM‐3 vascular activities. These results, together with our previous findings on the cardioprotective functions of CORM‐3, indicate that this molecule is an excellent prototype of water‐soluble CO carriers for studying the pharmacological and biological features of CO.

Bioactivity and Pharmacological Actions of Carbon Monoxide-Releasing Molecules

Carbon monoxide (CO) is a resourceful gas as recent advances in the area of cell signaling are revealing an unexpected physiological role for CO in the cardiovascular, immune and nervous systems. Transition metal carbonyls have been lately discovered to function as CO-releasing molecules (CO-RMs) and elicit distinct pharmacological activities in biological systems. Studies currently ongoing in our laboratories are investigating both the chemical and bioactive features of a series of water-soluble CO-RMs and their specific utilization as vasoactive mediators, anti-inflammatory agents and inhibitors of cellular and tissue damage. The data presented in this review corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO in mammals and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cellular and organ dysfunction.

VEGF enhances intraneural angiogenesis and improves nerve regeneration after axotomy

Whilst there is an increased understanding of the cell biology of nerve regeneration, it remains unclear whether there is a direct interrelationship between vascularisation and efficacy of nerve regeneration within a nerve conduit. To establish this is important as in clinical surgery peripheral nerve conduit grafting has been widely investigated as a possible alternative to the use of nerve autografts. The aim of this study was to assess whether vascular endothelial growth factor (VEGF), a highly specific endothelial cell mitogen, can enhance vascularisation and, indirectly, axonal regeneration within a silicone nerve regeneration chamber. Chambers containing VEGF (500–700 ng/ml) in a laminin‐based gel (Matrigel) were inserted into 1 cm rat sciatic nerve defects and nerve regeneration examined in relation to angiogenesis between 5 and 180 d. Longitudinal sections were stained with antibodies against endothelial cells (RECA‐1), axons (neurofilament) and Schwann cells (S‐100) to follow the progression of vascular and neural elements. Computerised image analysis demonstrated that the addition of VEGF significantly increased blood vessel penetration within the chamber from d 5, and by d 10 this correlated with an increase of axonal regeneration and Schwann cell migration. The pattern of increased nerve regeneration due to VEGF administration was maintained up to 180 d, when myelinated axon counts were increased by 78% compared with plain Matrigel control. Furthermore the dose‐response of blood vessel regeneration to VEGF was clearly reflected in the increase of axonal regrowth and Schwann cell proliferation, indicating the close relationship between regenerating nerves and blood vessels within the chamber. Target organ reinnervation was enhanced by VEGF at 180 d as measured through the recovery of gastrocnemius muscle weights and footpad axonal terminal density, the latter showing a significant increase over controls (P < 0.05). The results demonstrate an overall relationship between increased vascularisation and enhanced nerve regeneration within an acellular conduit, and highlight the interdependence of the 2 processes.