Antioxidants & Redox Signaling

This review focuses on the possible role of reactive oxygen species in the pathogenesis of this phenomenon. Evidence in support of a role of oxidants in preconditioning has come from the observation that administration of oxygen radical scavengers during the reperfusion period following the initial "preconditioning" ischemia could prevent the phenomenon. In addition, a brief exposure to a low, nontoxic dose of oxygen radicals may reproduce the beneficial effects of ischemic preconditioning, thus suggesting that radicals can directly trigger the preconditioning pathway. To explain the effects of oxidants in this setting, it has been suggested that reperfusion after the initial, "preconditioning" ischemic episode results in the generation of relatively low amounts of oxygen radicals, which are insufficient to determine cell necrosis, but nevertheless could modify cellular activities that have been implicated as mediators of the preconditioning phenomenon. Recent evidence suggests that low levels of oxidants may have a modulatory role on several cell functions. Possible mechanisms of oxidant-mediated protection might be protein kinase C and other kinases, ATP-dependent potassium channels, or changes in sulfhydryl group redox state, while an effect on adenosine metabolism, or the induction of myocardial stunning presumably does not contribute to oxidant-mediated preconditioning. Finally, de novo protein synthesis and gene expression, and increased antioxidant defenses might be involved in the late phase of preconditioning. In summary, available data strongly suggest that oxygen radicals might be possible mediators of preconditioning. However, further investigation is required to clearly elucidate their exact role and mechanisms of action.

Alterations in the production of nitric oxide (NO^•) are a critical factor in the injury that occurs in ischemic and reperfused myocardium; however, controversy remains regarding the alterations in NO^• that occur and how these alterations cause tissue injury. As superoxide generation occurs during the early period of reperfusion, the cytotoxic oxidant peroxynitrite (ONOO^-) could be formed; however, questions remain regarding ONOO^- formation and its role in postischemic injury. Electron paramagnetic resonance spin trapping studies, using the NO^• trap Fe²⁺-N-methyl-D-glucamine dithiocarbamate (Fe-MGD), and chemiluminescence studies, using the enhancer luminol, have been performed to measure the magnitude and time course of NO^• and ONOO^- formation in the normal and postischemic heart. Isolated rat hearts were subjected to control perfusion, or ischemia followed by reperfusion in the presence of Fe-MGD with electron paramagnetic resonance measurements performed on the effluent from these hearts. Whereas only trace signals were present prior to ischemia, prominent NO^• adduct signals were seen during the first 2 min of reflow. The reperfusion associated increase in these NO^• signals was abolished by nitric oxide synthase inhibition. In hearts perfused with luminol to detect ONOO^- formation, a similar marked increase was seen during the first 2 min of reperfusion that was blocked by nitric oxide synthase inhibitors and by superoxide dismutase. Either N ^G-nitro-L-arginine methyl ester or superoxide dismutase treatment resulted in more than twofold higher recovery of contractile function than in untreated hearts. Immunohistology studies demonstrated that the ONOO^--mediated nitration product nitrotyrosine was formed in postischemic hearts, but not in normally perfused controls. Thus, NO^• formation is increased during the early period of reperfusion and reacts with superoxide to form ONOO^-, which results in protein nitration and myocardial injury.

In this review, attempts were made to establish the role of reactive oxygen species as signaling molecules that regulate cardiomyocyte life and death during ischemia and reperfusion. Ischemia/reperfusion is a classical example because partial or mild ischemia can lead to simultaneous execution and repair of the cardiomyocytes, which is disrupted during severe ischemia leading to cell necrosis because of the lack of ATP. Apoptosis and repair processes are mediated by adaptive response in which oxygen free radicals function as typical signaling molecules through the activation of receptor tyrosine kinases, protein kinase C, and mitogen-activated protein kinases, as well as induction of redox-sensitive transcription factors and genes.

Protection of alveolar epithelial cells (alveolocytes) and vascular endothelial cells against pulmonary oxidative stress is an important problem. An inadequate delivery to the target cells limits the protective utility of the antioxidant enzymes, superoxide dismutase (SOD) and catalase. SOD and catalase modifications, such as coupling with polyethylene glycol and encapsulation in liposomes, prolong the life span of the active enzymes in vivo. The airway administration of SOD and catalase protects alveolocytes against hyperoxic oxidative stress. Although pulmonary endothelium is poorly accessible from the airways, it is accessible from circulation. However, antioxidant enzymes and their derivatives display poor targeting to pulmonary endothelium. To improve the targeting and provide intracellular delivery to endothelium, the enzymes can be conjugated with antibodies against endothelial antigens, such as angiotensin-converting enzyme and adhesion molecules [intercellular adhesion molecule-1 (ICAM-1) or platelet-endothelial cell adhesion molecule-1 (PECAM-1)]. These immunoconjugates accumulate in the pulmonary vasculature in intact animals, enter endothelium, and augment the antioxidant defenses. The immunoconjugates directed against ICAM-1 and PECAM-1 may also provide a secondary therapeutic benefit by blocking of sequestration and infiltration of leukocytes in the lungs. Further investigations are necessary to evaluate the therapeutic effectiveness of the vascular immunotargeting of antioxidant enzymes and solve technical problems associated with production of safe, clinically useful conjugates.

Oxygen-derived free radicals (O₂ ^•-, H₂O₂, and ^•OH) that are produced during postischemic reperfusion are currently suspected to be involved in the pathogenesis of tissue injury. Another reactive oxygen species, the electronically excited molecular oxygen (¹O₂), is of increasing interest in the area of experimental research in cardiology. In this review are discussed the main potential sources of singlet oxygen in the organism, particularly in the myocardium, the various cardiovascular cytotoxic effects induced by this reactive oxygen intermediate, and the growing evidence of its involvement in ischemia/reperfusion injury.

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H₂O₂ production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (non-apoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H₂O₂ synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.

Nitric oxide production via inducible nitric oxide synthase (iNOS) is believed to play a role in cardiac allograft rejection. Previously, we showed that antioxidants can significantly prolong cardiac graft survival, but the nature of this protection is unknown. In the present study, we examined the protective effect of another antioxidant, dimethylthiourea (DMTU), in a model of cardiac allograft rejection. Specifically, we hypothesized that DMTU would prolong graft survival and decrease activation of nuclear factor-κB (NF-κB), an important redox-sensitive transcription factor necessary for iNOS gene expression. NF-κB was activated by twofold as early as postoperative day 2 in allografts. NF-κB activation in allografts progressed to a peak of ninefold by postoperative day and remained increased until postoperative day 6. No activation of NF-κB was observed in isografts for comparable time periods. Treatment with DMTU resulted in a significant prolongation of graft survival. This beneficial effect was associated with diminished activation of myocardial NF-κB. Treatment with DMTU also resulted in decreased formation of iron-nitrosylprotein complexes as evidenced by electron paramagnetic resonance spectroscopy. These studies provide evidence that reactive oxygen plays a significant role in signal transduction for activation via the transcription factor, NF-κB, thereby modulating distal actions and consequences of iNOS-derived nitric oxide.

The process of angiogenesis is initiated primarily as a consequence of hypoxic stimulation at the cellular and molecular level. Although several angiogenic growth factors have been identified, at present a detailed understanding of the interplay among inducing stimuli, growth factors, and their respective molecular targets remains to be evaluated. Here we report the effects of progressively increasing durations of moderate hypoxia on the protein expression profiles and tissue distribution patterns of the vascular endothelial growth factor system and the angiopoietin/Tie system in the adult rat myocardium. The relative temporal trends of expression of the various components of these two systems, as well as apparent relationships between Flk-1 and angiopoietin-2 and between Flt-1 and Tie-1, suggest a probable sequence of involvement during myocardial angiogenesis, as proposed in our model. Such relationships may potentially be utilized in formulating strategies for sequential gene therapy to achieve clinically relevant myocardial angiogenesis.

Coenzyme Q₁₀ (CoQ) has long been utilized as a cardioprotective agent in various heart diseases. One of the most important mechanisms by which CoQ exerts cardioprotection is aerobic ATP production as a mobile electron carrier in the mitochondrial electron transfer chain. The ability of CoQ to afford myocardial protection is also attributed to its antioxidant property. However, CoQ may also act as a pro-oxidant through the generation of reactive oxygen species. Although excess oxidative stress is known to induce death signaling via cytochrome c release from mitochondria, it is now apparent that a brief exposure to oxidative stress stimulates redox signaling for acquisition of tolerance to oxidative stress. Therefore, we have investigated dual involvement of CoQ in redox signaling generation through enhanced production of reactive oxygen species and death signaling inhibition through antioxidation. Mitochondria were isolated from the rat heart and incubated with CoQ (10 or 100 μM) or its vehicle HCO 60 for 1 h. H₂O₂ and cytochrome c release from respiring mitochondria were increased by antimycin A (2 μM), an inhibitor of complex III respiratory chain, or by high Ca²⁺ (10 μM). This enhanced release of H₂O₂ was associated with an increase in lipid peroxidation as measured with 4-hydroxy-2-nonenal-modified proteins and with large amplitude swelling of mitochondria. CoQ potentiated H₂O₂ release from antimycin A- or high Ca²⁺-treated mitochondria, but was capable of inhibiting lipid peroxidation and large amplitude swelling, and attenuated cytochrome c release from the mitochondria. In addition, CoQ increased ATP synthesis by mitochondria. These results suggest that CoQ plays dual roles in mitochondrial generation of intracellular signaling. CoQ acts as a pro-oxidant that participates in redox signaling. CoQ also acts as an antioxidant that inhibits permeability transition and cytochrome c release, and increases ATP synthesis, thereby attenuating death signaling toward apoptosis and necrosis.

Protection of ischemic myocardium is an important unmet need in reperfusion therapy of acute myocardial infarction. Myocardial ischemia and reperfusion induce necrosis and apoptosis in cardiomyocytes. Caspase processing and activation are critical steps in most receptor and nonreceptor pathways of apoptosis. Caspase inhibitors have been shown to reduce ischemia reperfusion injury in cardiac muscle. Information about dose response and time of administration are needed to optimize the design of preclinical studies. We used isolated adult rabbit cardiomyocytes subjected to metabolic inhibition (MI) and recovery to examine the role of caspases and caspase inhibitors, the dose response, and the timing of administration. In vitro inhibitory concentrations (K _i) were determined for purified caspases. Cardiomyocytes subjected to MI were treated with peptidomimetic fluoromethyl ketone inhibitors of caspases before or during MI, or at recovery. Caspase inhibitors were most effective when added before MI and included throughout recovery, but were partially protective if added after MI. The optimal concentration of the inhibitors tested was ∼10 μM. Protection was sustained when cells were allowed to recover for 4 or 24 h. These results suggest that caspase activation is an important component of myocyte injury mediated by MI and recovery. Low doses of caspase inhibitors were identified that reduce injury in this model system, and further investigations using in vivo models are warranted.

We have assessed the relationship between reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO) mRNA expression using northern blotting, reverse transcription-polymerase chain reaction(RT-PCR), and enzyme activity in isolated working ischemic/reperfused rat hearts. Isolated hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Upon reperfusion with VF, cardiac function was registered (n = 6 in each group), and HO mRNAs and enzyme activities were measured at the end of reperfusion in hearts that showed VF or did not develop VF. The expression of HO-1 mRNA (about fourfold) was observed in ischemic/reperfused nonfibrillated myocardium in comparison with the nonischemic control hearts. In those hearts when VF was developed, the expression of HO-1 mRNA was not observed in comparison with the nonischemic control myocardium. The results measured by RT-PCR and enzyme analysis support the data obtained by northern blotting. In additional studies, we decided to approach the question from a different angle. Thus, the purpose of our work was also to study the role of HO expression and enzyme activity in electrically fibrillated hearts without the ischemic/reperfused protocol. To simulate the period of 10 min of reperfusion-induced VF, hearts were electrically fibrillated, then defibrillated, and perfused for an additional 110 min, and HO-1 mRNA expression and enzyme activities were determined. Thus, electrically induced VF resulted in about 60%, 60%, and 70% reduction in HO-1 mRNA expression, RT-PCR signal intensity, and enzyme activity, respectively, compared with the nonfibrillated ischemic/reperfused group. In conclusion, our data provide evidence that the development of reperfusion-induced VF inhibits HO-1 mRNA expression and enzyme activity in both electrically fibrillated myocardium and ischemic/reperfused fibrillated hearts. The results clearly show that HO-1 mRNA expression and enzyme activity were increased in ischemic/reperfused nonfibrillated myocardium, suggesting that interventions that are able to increase HO-1 mRNA expression and enzyme activity may prevent the development of VF.

The dose-dependent cardiomyopathy and heart failure due to adriamycin have been shown to be due to increased oxidative stress and loss of myocytes. We examined the incidence of myocardial apoptosis as well as changes in the expression of apoptotic regulatory gene products in an established animal model of adriamycin cardiomyopathy. Rats were treated with adriamycin (cumulative dose, 15 mg/kg), and the hearts were examined for apoptosis as well as expression of Bax, caspase 3, and Bcl-2 at 0, 4, 10, 16, and 21 days after the treatment. A significant increase in the incidence of apoptosis was seen at 4 days, followed by a decline at 10 and 16 days of posttreatment. At 21 days, the number of apoptotic cells increased again and included cells of the conducting system. Expression of Bax corresponded to these biphasic changes, whereas the converse was true for the expression of Bcl-2. The latter peaked at 10 days followed by a decline at 16 and 21 days. The Bax/Bcl-2 ratio also correlated with the incidence of apoptosis. Expression of caspase 3 correlated with increased apoptosis, but only at early time points. Probucol (cumulative dose, 120 mg/kg), a known antioxidant as well as promoter of endogenous antioxidants, significantly reduced the incidence of apoptosis as well as expression of Bax. Adriamycin-induced hemodynamic changes were also prevented by probucol. These data suggest that adriamycin-induced apoptosis is mediated by oxidative stress and may play a role in the development of heart failure.

Changes occur in gene expression during aging in vivo and in replicative senescence in vitro, suggesting that aging can affect gene regulation. We have recently observed age-related changes in ubiquitously expressed, oxidative stress-responsive nuclear factor-κB (NF-κB) pathway during aging. Here we report a significant age-related increase in nuclear NF-κB binding activity together with increased protein levels of p52 and p65 components in rat liver. An additional, higher molecular weight protein band seen in their western blots suggests that their post-translational modification(but not phosphorylation) occurs in liver, which might affect their nuclear localization and binding activity during aging. However, aging did not affect the protein levels of the main IκB inhibitors(IκBα and IκBβ) or IκB kinase (IKK)-complex subunits (IKKα, -β, and -γ) involved in NF-κB activation. In addition, the level of Ser³²-phosphorylated IκBα was unaffected by age, suggesting that neither the IKK complex nor altered level of the main inhibitors is involved in the observed up-regulation of NF-κB binding activity. Furthermore, the expression of NF-κB mRNAs (p50, p52, p65, and c-rel) and the mRNAs of their inhibitors (IκBα and IκBβ) did not show any statistically significant age-related changes. These results indicate that the expression level of NF-κB genes is not significantly affected by aging. The up-regulation of constitutive nuclear NF-κB binding activity and increased levels of nuclear p52 and p65 proteins might affect the expression of some NF-κB target genes in the aging liver.

Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H₂O₂, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 ± 39 μM H₂O₂ in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 ± 17 μM H₂O₂ is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H₂O₂ at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H₂O₂ at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.

Peroxynitrite, formed by the combination of superoxide anion and nitric oxide, is a powerful oxidant at physiological pH and is apparently involved in the pathogenesis of several human diseases. Therefore, inhibitors of peroxynitrite-induced oxidation are important targets for pharmaceutical development. The reaction of peroxynitrite with L-tyrosine, one of its biological targets, yields stable products, including nitrotyrosine and dityrosine. Here we test the ability of thiols, nitric oxide donors, and purine derivatives to inhibit peroxynitrite-induced dityrosine formation in a physiological buffer containing bicarbonate/CO₂. We show that both reduced and oxidized thiols, nitric oxide donors, and urate, but not other purine derivatives, reduce peroxynitrite-induced dityrosine formation.