HO-1 induction restores c-AMP-dependent lung epithelial fluid transport following severe hemorrhage in rats.

Inhibition of cAMP-dependent stimulation of the vectorial fluid transport across the lung epithelium following hemorrhagic shock is mediated by NO released within the airspaces of the lung. We tested here the hypothesis that prior induction of HO-1 would attenuate the release of NO in the airspaces, thus preventing the inhibition of the c-AMP stimulation of alveolar fluid clearance (ALC) in rats. Indeed, HO-1 induction restored the cAMP-mediated up-regulation of ALC after hemorrhage by decreasing NO released within the airspaces of the lung. In vitro studies demonstrated that HO-1 induction significantly reduced the iNOS-mediated release of NO by alveolar macrophages stimulated with endotoxin for 24 h. This effect is explained in part by a HO-1-dependent attenuation of the LPS-mediated nuclear translocation of NF-kappaB. In addition, HO-1 induction also significantly reduced the iNOS-mediated release of NO by MH-S cells that were stimulated with interferon-gamma by decreasing the phosphorylation of STAT 1, another transcription factor important for the activation of the iNOS promoter. In contrast, HO-1 induction did not affect the production of NO by rat alveolar epithelial type II cells that were stimulated with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) for 24 h. In summary, these results provide the first in vivo evidence that the induction of HO-1 in the lung restores a normal fluid transport capacity of the alveolar epithelium following hemorrhagic shock by inhibiting the iNOS-mediated release of NO by alveolar macrophages.

Unique effects of zinc protoporphyrin on HO-1 induction and apoptosis.

Zinc protoporphyrin (ZnPP), a naturally occurring molecule, is increased in iron deficiency and lead intoxication. ZnPP can also induce heme oxygenase (HO-1), the enzyme it competitively inhibits. In cultured cells (HA-1), ZnPP was the strongest HO-1 inducer of any metalloporphyrin (MP) tested. This was not due to increased oxidative stress, enhanced binding at metal response element, nor increased binding at activator protein-1 (AP-1) or SP-1 sites on HO-1. Only ZnPP, however, increased binding of nuclear proteins to early growth response-1 (Egr-1) protein consensus sequence. Pretreatment of HA-1 with cycloheximide inhibited ZnPP-induced HO-1 messenger RNA (mRNA) by 55%. Incubation with antisense Egr-1 oligomers decreased ZnPP-induced HO-1 expression by 47%. Furthermore, the level of HO-1 mRNA induction by ZnPP was 2-fold less in Egr-1-deficient fibroblasts than in wild-type cells. Because no Egr-1 binding site was previously identified on the HO-1 promoter, HA-1 cells were transfected with HO-1 CAT constructs containing segments of a 12.5-kb enhancer region of HO-1. A 196-bp fragment (RH) located approximately 9.5 kb upstream of the transcription start site mediated HO-1 induction by ZnPP alone. DNase I footprinting analysis further revealed that nuclear proteins bound to a 50-bp sequence in the RH. Within this sequence, a novel 9-bp region with 78% homology to the Egr-1 consensus sequence was identified further suggesting that Egr-1 partially mediates HO-1 induction by ZnPP. Lastly, increased apoptosis and nuclear localization were only seen with ZnPP, suggesting that increased ZnPP in disease states may serve as a cellular signaling mechanism.

The biology of bilirubin production.

Heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, has been identified from the late 1960s. This enzyme has been shown to have many other roles in recent years. The inducible form is regulated by oxidative stress, inflammation, and heavy metals, among others, and is cytoprotective in many instance. Nonetheless, there are instances when HO-1 can be deleterious due to the release of iron from the reaction. Another important by-product, carbon monoxide, is a vasodilator and a neurotransmitter and has been implicated in signal transduction pathways. More recently, nonenzymatic, signaling roles of HO have been suggested. This may serve to regulate the endogenous activity of this enzyme when cellular heme levels are low.

Synergistic inhibition of cyclooxygenase-2 expression by vitamin E and aspirin.

The use of aspirin in rheumatoid arthritis is limited since inhibition of the pro-inflammatory enzyme cyclooxygenase-2 occurs only at higher aspirin doses that are often associated with side effects such as gastric toxicity. Using a macrophage cell line (J774. 1A), the present study explores possible synergistic effects of aspirin and vitamin E on the expression and activity of cyclooxygenase-2. Lipopolysaccharide-induced prostaglandin E(2) formation was significantly reduced by aspirin (1-100 microM) or vitamin E (100-300 microM). When combined with vitamin E, aspirin-dependent inhibition of prostaglandin E(2) formation was increased from 59% to 95% of control. Likewise, lipopolysaccharide-induced cyclooxygenase-2 protein and mRNA expression were virtually abolished by the combined treatment of aspirin and vitamin E, whereas the two agents alone were only modestly effective. Vitamin C did not mimic the actions of vitamin E under these conditions, suggesting that redox-independent mechanisms underlie the action of vitamin E. In agreement with this, vitamin E and aspirin were without effect on lipopolysaccharide-induced translocation of the redox-sensitive transcription factor NF-kappa B. Our results show that co-administration of vitamin E renders cyclooxygenase-2 more sensitive to inhibition by aspirin by as yet unknown mechanisms. Thus, anti-inflammatory therapy might be successful with lower aspirin doses when combined with vitamin E, thereby possibly avoiding the side effects of the usually required high dose aspirin treatment.

End-tidal carbon monoxide measurements in women with pregnancy-induced hypertension and preeclampsia.

OBJECTIVE: We sought to compare the end-tidal carbon monoxide breath levels in pregnant women with and without pregnancy-induced hypertension and preeclampsia. STUDY DESIGN: We prospectively performed end-tidal carbon monoxide measurements corrected for ambient carbon monoxide in nonsmoking women during late gestation (>31 weeks). The study group included 22 women with pregnancy-induced hypertension or symptoms of preeclampsia and a control group of 20 normotensive pregnant women. RESULTS: The carbon monoxide measurements corrected for ambient carbon monoxide (mean +/- SD) were significantly lower (P <.01) in the hypertensive group than in the control group (1.17 +/- 0.35 vs 1.70 +/- 0.54 ppm). The study group had a significantly higher number of low (<1.2 ppm) end-tidal carbon monoxide measurements corrected for ambient carbon monoxide (13 [59.1%] vs 1 [5.0%]; P <.001). The end-tidal carbon monoxide measurements corrected for ambient carbon monoxide remained significantly lower in comparison with those found in the control group when the study group was divided into women with pregnancy-induced hypertension only (n = 11) and those with preeclampsia (n = 11) (1.19 +/- 0.37 ppm; P <.01; and 1.15 +/- 0.41 ppm; P <.01; respectively). CONCLUSIONS: Our findings suggest that carbon monoxide formation may be significantly lower in women with pregnancy-induced hypertension and preeclampsia. These data suggest that carbon monoxide could have a contributory role in the apparent paradox of the seemingly protective effect of smoking to decrease the risk of preeclampsia.

Carbon monoxide detection and biological investigations.

Even though the heme degradation pathway consists of only two reactions, it and its major enzyme (i.e. HO), nonetheless, impact other processes not only through the removal of excess heme, but also through the production of several metabolically active compounds. Thus CO and biliverdin along with reactive iron, Fe2, are the primordial products of this ancient, highly conserved reaction. That every component of the heme catabolic pathway is directly or indirectly related to other reactions involving oxygen or light is, perhaps, no accident of nature. That a fundamentally destructive event can be linked with a multiplicity of synthetic events and various biological effects, depending on the timing and location of the HO activity, is testament to the economy and the ultimate beauty of nature. Furthermore, the interaction of the heme catabolic pathway with that of the NOS system may lead to even more exciting avenues of research. It may be shown that the integrity of the heme catabolic pathway, which is ever present and plays a role in every tissue, is central to the existence of most complex organisms.

HO-1 expression in type II pneumocytes after transpulmonary gene delivery.

Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.

Regulation and role of heme oxygenase in oxidative injury.

The HO-1 isoenzyme is an early stress response gene regulated by many forms of oxidative stress. The HO-2 isoenzyme is predominantly a constitutive enzyme, which may serve to sequester heme as well as degrade it. All HO enzyme activity results in the degradation of heme and the production of antioxidant bile pigments, which would favor an antioxidant role for the enzyme. In fact, in oxidative stress in vitro, HO-1 is protective (91-94) but within a narrow threshold of overexpression (93,94) in some models, since iron released in the HO reaction may obviate any cytoprotective effect (Fig. 3). So far, HO-2 appears to be beneficial in oxygen toxicity in vivo, but the consequences of HO-2 overexpression have not yet been tested. It will be important to better define the role of each HO isoenzyme in oxidative stress so as to determine whether enhancing these complex systems could alleviate some of the cellular changes seen as a result of oxidative injury. Furthermore, prior to considering therapeutic maneuvers to enhance HO, a complete understanding of the physiologic consequences of HO-1 induction and associated reactions, in each particular setting, will be crucial.

A role for oxidative stress in suppressing serum immunoglobulin levels in lead-exposed Fisher 344 rats.

Evidence implicating oxidative stress in toxicity during lead intoxication in vivo has opened new avenues for investigation of the mechanisms of lead-induced immunosuppression. The current study explores the possibility that lead-induced oxidative stress contributes to the immunosuppression observed during lead poisoning. Fisher 344 rats were exposed to 2,000 ppm lead acetate in their drinking water for 5 weeks. One week following removal of lead from the drinking water, significant reductions in serum levels of IgA, IgM, and IgG were detected. Significant increases in oxidative damage, based on malondialdehyde (MDA) content, were observed in peripheral blood mononuclear cells (PMCs) collected during the same experiments. In addition, MDA content increased in livers from lead-exposed rats. Following 5 weeks of lead exposure, administration of either 5.5 mmol/kg N-acetylcysteine (NAC) or 1 mmol/kg meso-2,3-dimercaptosuccinic acid (DMSA) in the drinking water for 1 week significantly reversed the inhibitory effects of lead on serum immunoglobulin (Ig) levels. Also, all parameters indicative of oxidative stress returned to control levels. These results suggest that oxidative stress contributes to suppressed serum Ig levels during lead intoxication in vivo, and that intervention with either a thiol antioxidant (NAC) or a metal chelator (DMSA) will alleviate this lead-induced suppression by correcting the prooxidant/antioxidant imbalance caused by lead exposure.

Heme oxygenase-1 is a cGMP-inducible endothelial protein and mediates the cytoprotective action of nitric oxide.

Inducible heme oxygenase (HO-1) has recently been recognized as an antioxidant and cytoprotective gene. By use of Western blotting, cell viability analysis, and antisense technique, the present study investigates the involvement of HO-1 in endothelial protection induced by the clinically used nitric oxide (NO) donor molsidomine (specifically, its active metabolite 3-morpholinosydnonimine [SIN-1]) and the second messenger cGMP. In bovine pulmonary artery endothelial cells, SIN-1 and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 to 100 micromol/L induced the synthesis of HO-1 protein in a concentration-dependent fashion up to 3-fold over basal levels. HO-1 induction by SIN-1 was inhibited in the presence of the NO scavenger phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4, 3-a]quinoxalin-1-one. 8-Bromo-cGMP (1 to 100 micromol/L) and dibutyryl cGMP (1 to 100 micromol/L) as well as the activator of particulate guanylyl cyclase atrial natriuretic peptide (1 to 100 nmol/L) produced increases in HO-1 protein similar to those produced by SIN-1. SIN-1 and 8-bromo-cGMP increased heme oxygenase activity (bilirubin formation). Cytoprotection by NO donors was abrogated in the presence of the heme oxygenase inhibitor tin protoporphyrin IX. Pretreatment of cells with a phosphorothioate-linked HO-1 antisense oligonucleotide prevented protection by SIN-1 or 8-bromo-cGMP against tumor necrosis factor-alpha cytotoxicity, whereas sense and scrambled HO-1 were without effect under these conditions. Our results show for the first time that HO-1 is a cGMP-sensitive endothelial gene and establish conclusively a causal relationship between HO-1 induction and endothelial protection by the NO/cGMP system. By targeting cytoprotective HO-1, NO donors may therefore be expected to induce antioxidant, antiatherogenic, and anti-inflammatory effects.

Maturational differences in hyperoxic AP-1 activation in rat lung.

Immature organisms (neonates; <12 h old) have vastly differing responses to hyperoxic injury than adults. A common feature of hyperoxic gene regulation is involvement of activator protein (AP)-1. We evaluated lung AP-1 binding as well as that of the AP-1 subunit proteins c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D after exposure to >95% O(2) for 3 days. Unlike adults, neonates showed no increased AP-1 binding in hyperoxia despite a high affinity of the AP-1 binding complexes for phosphorylated c-Jun and Jun D as demonstrated by supershift of these antibodies with the AP-1 complexes. Moreover, neonatal lungs exhibited two distinguishable AP-1 binding complexes, whereas adult lungs had one. In neonates, sequential immunoprecipitation revealed that the lower AP-1 complex was composed of proteins from both the Fos and Jun families, whereas the upper complex consisted of Jun family proteins, with predominance of Jun D. In adults, the single AP-1 complex appeared to involve other Fos or non-Fos or non-Jun family proteins as well. Neonatal lungs showed a higher level of Jun B and Jun D immunoreactive proteins in both air and hyperoxia compared with those in adult lungs. These results suggest that significant maturational differences in lung AP-1 complexes exist and that these may explain transcriptional differences in hyperoxic gene regulation.

Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.

It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.

Loss of the ataxia-telangiectasia gene product causes oxidative damage in target organs.

Ataxia-telangiectasia (A-T) is characterized by a markedly increased sensitivity to ionizing radiation, increased incidence of cancer, and neurodegeneration, especially of the cerebellar Purkinje cells. Ionizing radiation oxidizes macromolecules and causes tissue damage through the generation of reactive oxygen species (ROS). We therefore hypothesized that A-T is due to oxidative damage resulting from loss of function of the A-T gene product. To assess this hypothesis, we employed an animal model of A-T, the mouse with a disrupted Atm gene. We show that organs which develop pathologic changes in the Atm-deficient mice are targets of oxidative damage, and that cerebellar Purkinje cells are particularly affected. These observations provide a mechanistic basis for the A-T phenotype and lay a rational foundation for therapeutic intervention.

Antioxidant and cytotoxic effects of bilirubin on neonatal erythrocytes.

Bilirubin, the breakdown product of heme from erythrocytes, accumulates in the neonate in the first days of life. In recent years, the antioxidant properties of bilirubin have been demonstrated in vitro and in vivo, yet it is clear that bilirubin can be toxic to cells. To study the range in which bilirubin exerts its beneficial effect, we used erythrocytes derived from cord blood and incubated them with 0-60 mg/dL bilirubin combined with 3 g/dL BSA (bilirubin/BSA) to mimic physiologic and pathologic conditions. Oxidative stress was induced by incubating the erythrocytes with a solution of 0.6 mM H2O2 and 0.15 M CuSO4 to generate hydroxyl radical mediated injury. The loss of fluorescence of cis-parinaric acid and the degree of protein oxidation of erythrocyte membranes were assessed. Additionally, we determined erythrocyte membrane integrity, glucose-6 phosphate dehydrogenase activity, and adenosine triphosphatase activity before and after incubation with bilirubin/BSA. Incubation with bilirubin/BSA at concentrations up to 60 mg/dL and a bilirubin/BSA molar ratio of two was associated with dose-dependent protection of erythrocytes against lipid peroxidation. However, concentrations of bilirubin equal to or exceeding 30 mg/dL and a bilirubin:BSA ratio of one were associated with increased protein oxidation, decreased erythrocyte glucose-6 phosphate dehydrogenase and adenosine triphosphatase activity, and altered cell membrane integrity. We conclude that bilirubin, at physiologic concentrations, protects neonatal red blood cells against oxidative stress in the presence of physiologic concentrations of BSA but that bilirubin concentrations of 30 mg/dL or higher and a bilirubin:BSA ratio of greater than one are associated with significant cytotoxicity.

Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells.

Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.

Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2.

Heme oxygenase (HO) activity leads to accumulation of the antioxidant bilirubin, and degradation of the prooxidant heme. Moderate overexpression of the inducible form, HO-1, is associated with protection against oxidative injury. However, the role of HO-2 in oxidative stress has not been explored. We evaluated survival, indices of oxidative injury, and lung and HO expression in HO-2 null mutant mice exposed to > 95% O2 compared with wild-type controls. Similar basal levels of major lung antioxidants were observed, except that the knockouts had a twofold increase in total glutathione content. Despite increased HO-1 expression from HO-1 induction, knockout animals were sensitized to hyperoxia-induced oxidative injury and mortality, and also had significantly increased markers of oxidative injury before hyperoxic exposure. Furthermore, during hyperoxia, lung hemoproteins and iron content were significantly increased without increased ferritin, suggesting accumulation of available redox-active iron. These results demonstrate that the absence of HO-2 is associated with induction of HO-1 and increased oxygen toxicity in vivo, apparently due to accumulation of lung iron. These results suggest that HO-2 functions to augment the turnover of lung iron during oxidative stress, and that this function does not appear to be compensated for by induction of HO-1 in the knockouts.