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Heme oxygenase-1 (HO-1) is a redox-sensitive inducible protein that provides efficient cytoprotection against oxidative stress. Curcumin, a polyphenolic natural compound that possesses anti-tumor and anti-inflammatory properties, has been reported recently to induce potently HO-1 expression in vascular endothelial cells (Free Rad Biol Med28:1303–1312, 2000). Here, we extend our previous findings by showing that caffeic acid phenethyl ester (CAPE), another plant-derived phenolic agent, markedly increases heme oxygenase activity and HO-1 protein in astrocytes. The effect seems to be related to the peculiar chemical structures of curcumin and CAPE, because analogous antioxidants containing only portions of these two molecules were totally ineffective. At a final concentration of 30 μM, both curcumin and CAPE maximally up-regulated heme oxygenase activity while promoting marked cytotoxicity at higher concentrations (50–100 μM). Similar results were obtained with Curcumin-95, a mixture of curcuminoids commonly used as a dietary supplement. Incubation of astrocytes with curcumin or CAPE at concentrations that promoted maximal heme oxygenase activity resulted in an early increase in reduced glutathione followed by a significant elevation in oxidized glutathione contents. A curcumin-mediated increase in heme oxygenase activity was not affected by the glutathione precursor and thiol donor N-acetyl-l-cysteine. These data suggest that regulation of HO-1 expression by polyphenolic compounds is evoked by a distinctive mechanism which is not necessarily linked to changes in glutathione but might depend on redox signals sustained by specific and targeted sulfydryl groups. This study identifies a novel class of natural substances that could be used for therapeutic purposes as potent inducers of HO-1 in the protection of tissues against inflammatory and neurodegenerative conditions.

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2)

The expression of the inducible haem oxygenase (HO‐1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO‐1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO‐1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO‐1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO‐1 gene expression follows a fibre type‐specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.

To study the mechanism by which cell-free hemoglobin preparations may alter coronary vascular reactivity, we investigated the effect of human hemoglobin cross-linked between alpha chains with bis(3,5-dibromosalicyl)fumarate (ααHb) on the vasomotor response to acetylcholine (ACh) in isolated perfused rabbit hearts. Dose-response curves were generated by monitoring the increase in coronary pressure during serial addition of 0.2-10 μM ACh before, during and after 20 min infusion of three test solutions: a) 0.1 g/dl ααHb (62 μM heme); b) 0.1 g/dl ααHb plus 60 μM deferoxamine (DFO); c) 50 μM NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide (NO) synthase. We found that the sensitivity to ACh-induced vasoconstriction was significantly potentiated in the presence of ααHb and L-NAME. In addition, this response was only partially reversed after removal of ααHb, except when DFO was simultaneously infused with the ααHb solution. These findings are consistent with the idea that both NO binding to hemoglobin and iron-mediated oxygen free radical generation contribute to an altered coronary vasomotor responsiveness induced by cell-free hemoglobin.

The xanthine/xanthine oxidase reaction produces reproducible amounts of oxygen-derived free radicals that oxidize human oxyhemoglobin (Hb). We monitored the kinetics of the oxidation of stripped Hb (sHb), purified HbA0 and α-α cross-linked Hb (HbXL99α) at [Hb] in the 5 to 150 μM (heme) range. For increasing [Hb], the oxidation halftime (t1/2) increased for all Hbs, but t1/2 was always less for HbXL99α than for HbA0 and sHb. Such feature was attributed to the lower affinity for O2 of HbXL99α and may represent a serious problem for use of this Hb as blood substitute.

Hypoxanthine is the final product of the catabolism of ATP in the stored red cell. Upon transfusion, this purine may be uptaken by the endothelial cell and oxidized in a post-ischemic or post-anoxic environment with production of oxygen-derived free radicals. We have tested this hypothesis with a isolated perfused rat heart model monitoring the recovery of the heart function from 20 min anoxia in the presence of 0.1 mM hypoxanthine or xanthine. Addition of 0.1 mM guanine minimized the fraction of hypoxanthine to be salvaged. The presence of hypoxanthine in the vascular space impaired the recovery of the end-diastolic pressure, left ventricular developed pressure, contraction rate, and coronary perfusion pressure. We conclude that intravascular hypoxanthine is oxidized by the endothelial cell xanthine oxidase contributing to the post-anoxic reoxygenation injury. Since the injury led by equimolar xanthine was nearly half of that observed for hypoxanthine, this injury appears to be correlated to the stoichiometry of the oxygen-derived free radical generating reaction.

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) and S-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 of S-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.

Evidence has been put forth that a number of human and experimental cardiomyopathies are associated with a lower myocardial carnitine content. This study was performed to test the hypothesis that the correction of carnitine deficiency by a naturally occurring carnitine derivative, propionyl-L-carnitine (PLC), may improve cardiac function. Repeated administration of PLC was compared to saline with respect to cardiac function in rats with pressure-overload cardiac hypertrophy and low myocardial carnitine levels. Cardiac hypertrophy was induced by abdominal aorta constriction in rats. Separate groups of rats were used for (a) determination of myocardial carnitine content, (b) evaluation of in vivo hemodynamics, and (c) evaluation of performance and metabolic state of Langendorff perfused hearts. Results showed the following: (i) The myocardial carnitine content was inversely correlated to cardiac hypertrophy (r= 0.68, p < 0.05) and PLC treatment (50 mg/kg i.a. for 4 days) restored it to normal values (ii) The PLC effect on cardiac function was significantly and directly related to cardiac hypertrophy [correlations between heart weight and percent changes in cardiovascular parameters: cardiac output (CO), p < 0.001; cardiac work (CW), p < 0.01, stroke volume (SV) and stroke work (SW), p < 0.02]. In animals with heart weight > 1,400 mg, the effect of PLC on CO, CW, SV, SW, and total peripheral resistance (TPR) was significantly different from that of saline (CO, CW, SV, and SW, p < 0.005 each; TPR, p < 0.05). The effect was observed 24 h after the first PLC administration and significantly diminished following a 4 day suspension of the treatment, (iii) Perfused hearts from PLC-treated rats displayed a significantly lower left ventricular end-diastolic pressure (p < 0.01) and greater relaxation rate (p < 0.05) than those from control rats. Moreover, in PLC-treated hearts, the content of creatine phosphate, ATP, and total adenine nucleotides (ATP + ADP + AMP; TAN) was significantly increased (CP, p < 0.05; ATP and TAN, p < 0.01 vs. control). These data show that PLC exerts a stimulatory activity on hearts with hypertrophy and low carnitine content, implying that carnitine deficiency may contribute to the depression of cardiac function in this model.

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