The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Role of Heme Oxygenase-1 in Dual Stress Response of Herbicide and Micronutrient Fe in Arabidopsis thaliana.

Increasingly intensive agricultural practices are leading not only to herbicide contamination but also to nutritional stress on nontarget plants. This study evaluated the role of heme oxygenase-1 (HO-1) in the dual stress response of herbicide dichlorprop and micronutrient Fe in Arabidopsis thaliana. Our results revealed that co-treatment with 20 µM zinc protoporphyrin (a specific inhibitor of HO-1) reduced the activity of HO-1 by 21.6%, Fe2+ content by 19.8%, and MDA content by 20.0%, reducing abnormal iron aggregation and oxidative stress in response to the herbicide compared to treatment with (R)-dichloroprop alone, which has herbicidal activity. Thus, free Fe2+ released from HO-1 mediated dichlorprop-induced oxidative stress in the Fenton reaction and affected aberrant Fe aggregation, which also had an enantioselective effect. This study contributes to an in-depth understanding of the toxicity mechanism of herbicides under nutrient stresses, thus providing new strategies to control the environmental risks of herbicides.

Anti-Photoaging Effect of Hydrolysates from Pacific Whiting Skin via MAPK/AP-1, NF-κB, TGF-ß/Smad, and Nrf-2/HO-1 Signaling Pathway in UVB-Induced Human Dermal Fibroblasts.

Chronic exposure to ultraviolet (UV) light promotes the breakdown of collagen in the skin and disrupts the extracellular matrix (ECM) structure, leading to skin wrinkling. Pacific whiting (Merluccius productus) is a fish abundant on the Pacific coast. In the current study, we investigated the anti-wrinkle effect of hydrolysate from Pacific whiting skin gelatin (PWG) in UVB-irradiated human dermal fibroblasts and the molecular mechanisms involved. PWG effectively restored type 1 procollagen synthesis reduced by UVB-irradiation. Also, we found that PWG inhibited collagen degradation by inhibiting MMP1 expression. Furthermore, PWG decreased cytokines TNF-α, IL-6, and IL-1ß associated with inflammatory responses and increased antioxidant enzymes, HO-1, SOD, GPx, CAT, and GSH content, a defense system against oxidative stress. In terms of molecular mechanisms, PWG increased collagen synthesis through activating the transforming growth factor ß (TGF-ß)/Smad pathway and decreased collagen degradation through inhibiting the mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) pathway. It also suppressed the inflammatory response through suppressing the nuclear factor-κB (NF-κB) pathway and increased antioxidant enzyme activity through activating the nuclear factor erythroid 2/heme oxygenase 1 (Nrf-2/HO-1) pathway. These multi-target mechanisms suggest that PWG may serve as an effective anti-photoaging material.

Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration.

Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that cause loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate that zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.

Curcumin and Heme Oxygenase: Neuroprotection and Beyond.

Curcumin is a natural polyphenol component of Curcuma longa Linn, which is currently considered one of the most effective nutritional antioxidants for counteracting free radical-related diseases. Several experimental data have highlighted the pleiotropic neuroprotective effects of curcumin, due to its activity in multiple antioxidant and anti-inflammatory pathways involved in neurodegeneration. Although its poor systemic bioavailability after oral administration and low plasma concentrations represent restrictive factors for curcumin therapeutic efficacy, innovative delivery formulations have been developed in order to overwhelm these limitations. This review provides a summary of the main findings involving the heme oxygenase/biliverdin reductase system as a valid target in mediating the potential neuroprotective properties of curcumin. Furthermore, pharmacokinetic properties and concerns about curcumin's safety profile have been addressed.

The heme oxygenase system selectively enhances the anti-inflammatory macrophage-M2 phenotype, reduces pericardial adiposity, and ameliorated cardiac injury in diabetic cardiomyopathy in Zucker diabetic fatty rats.

Cardiac function is adversely affected by pericardial adiposity. We investigated the effects of the heme oxygenase (HO) inducer, hemin on pericardial adiposity, macrophage polarization, and diabetic cardiopathy in Zucker diabetic fatty rats (ZDFs) with use of echocardiographic, quantitative real-time polymerase chain reaction, Western immunoblotting, enzyme immunoassay, and spectrophotometric analysis. In ZDFs, hemin administration increased HO activity; normalized glycemia; potentiated insulin signaling by enhancing insulin receptor substrate 1(IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB)/Akt; suppressed pericardial adiposity, cardiac hypertrophy, and left ventricular longitudinal muscle fiber thickness, a pathophysiological feature of cardiomyocyte hypertrophy; and correspondingly reduced systolic blood pressure, total peripheral resistance, and pro-inflammatory/oxidative mediators, including nuclear factor κB (NF-κB), cJNK, c-Jun-N-terminal kinase (cJNK), endothelin (ET-1), tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1ß, activating protein 1 (AP-1), and 8-isoprostane, whereas the HO inhibitor, stannous mesoporphyrin, nullified the effects. Furthermore, hemin reduced the pro-inflammatory macrophage M1 phenotype, but enhanced the M2 phenotype that dampens inflammation. Because NF-κB activates TNFα, IL-6, and IL-1ß and TNF-α, cJNK, and AP-1 impair insulin signaling, the high levels of these cytokines in obesity/diabetes would create a vicious cycle that, together with 8-isoprostane and ET-1, exacerbates cardiac injury, compromising cardiac function. Therefore, the concomitant reduction of pro-inflammatory cytokines and macrophage infiltration coupled to increased expressions of IRS-1, PI3K, and PKB may account for enhanced glucose metabolism and amelioration of cardiac injury and function in diabetic cardiomyopathy. The hemin-induced preferential polarization of macrophages toward anti-inflammatory macrophage M2 phenotype in cardiac tissue with concomitant suppression of pericardial adiposity in ZDFs are novel findings. These data unveil the benefits of hemin against pericardial adiposity, impaired insulin signaling, and diabetic cardiomyopathy and suggest that its multifaceted protective mechanisms include the suppression of inflammatory/oxidative mediators.

Carbon monoxide: a vital signalling molecule and potent toxin in the myocardium.

Endogenous carbon monoxide (CO) is generated through the heme oxygenase-catalysed degradation of heme and is now established as an important, biologically active molecule capable of modulating a number of signalling pathways. Such pathways include those involving nitric oxide/guanylate cyclase, reactive oxygen species (ROS) and MAP kinases. In the heart, up-regulation of the inducible form of heme oxygenase (HO-1) following stresses such as ischemia/reperfusion provides cardioprotection, and much evidence indicates that CO accounts for many of these beneficial effects. One target of CO appears to be the L-type Ca(2+) channel; CO inhibits recombinant and native forms of this cardiac channel via mitochondria-derived ROS, which likely contributes to the protective effects of CO. In stark contrast, exposure to exogenous CO is toxic: chronic, low-level exposure can lead to myocardial injury and fibrosis, whereas acute exposure is associated with life-threatening arrhythmias. The molecular mechanisms accounting for such effects remain to be elucidated, but require future study before the potentially beneficial effects of CO therapy can be safely exploited. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Papel de la hemooxigenasa-1 en las enfermedades neurodegenerativas / Role of hemeoxygenase-1 in the neurodegenerative disorders

Objetivo. Revisar evidencias de la participación de la hemooxigenasa-1 (HO-1) en enfermedades neurodegenerativas. Desarrollo. La HO cataliza la degradación del grupo hemo a monóxido de carbono, hierro y biliverdina. Se han caracterizado ampliamente dos isoformas de la HO: una inducible (HO-1) y una constitutiva (HO-2). Como la expresión de HO-1 confiere citoprotección en varias líneas celulares y en modelos animales bajo estrés oxidativo, se considera que la activación del gen de HO-1 es un mecanismo de defensa celular. En estudios post mortem en cerebro se ha encontrado un incremento en la expresión de la HO-1 en pacientes con enfermedades de Alzheimer, Parkinson y Huntington. Aunque no se han determinado la causa y el significado de este aumento, existen evidencias de que la sobreexpresión de la HO-1 contribuye a la acumulación de hierro en la mitocondria, lo que sugeriría que la expresión de la HO-1 tiene un efecto citotóxico. En contraste, hay evidencias de que la sobreexpresión de la HO-1 disminuye la muerte celular en ratones transgénicos y cultivos neuronales expuestos a compuestos neurotóxicos, lo que sugeriría que esta enzima tiene un papel citoprotector. Conclusión. Existe controversia sobre si la expresión de la HO-1 durante enfermedades neurodegenerativas confiere citoprotección o, por el contrario, promueve la neurodegeneración. Por tanto, es necesario continuar el estudio del papel de la HO-1 en modelos de daño neuronal

Aim. To review some evidences about the role of hemeoxygenase-1 (HO-1) in neurodegenerative disorders. Development. HO is the rate-limiting enzyme that catalyzes the conversion of heme into biliverdin, carbon monoxide, and free iron. They are the inducible HO-1 and the constitutive HO-2. A large body of evidence suggests that HO-1 confers cytoprotection against oxidative stress. Postmortem studies conducted in humans have revealed increase in HO-1 protein in association with Alzheimer disease, Parkinson disease and Huntington disease. It is unknown the meaning of that increase. Nevertheless, there are evidences indicating that the overexpression of HO-1 contributes to the pathological iron deposition suggesting a detrimental role of HO-1. In contrast, there are evidences indicating that the overexpression of HO-1 decreases the neurotoxin-induced cell death in transgenic mice and neuronal cultures suggesting a cytoprotective role of HO-1. Conclusion. It is controversial if the overexpression of HO-1 has a detrimental or cytoprotective role. Therefore, it is necessary to continue the study about the role of the HO-1 in neurodegenerative diseases

Carbon dioxide insufflation attenuates parietal blood flow obstruction in distended colon: potential advantages of carbon dioxide insufflated colonoscopy.

BACKGROUND: Bowel distention after colonoscopy has been considered as a cause of blood flow disturbance. Carbon dioxide (CO2), with its higher absorbability and vasodilating effect, may reduce parietal blood flow disturbance of distended colon when used for intraluminal insufflation instead of air. The purpose of this study was to assess parietal blood flow of the colon distended with intraluminal air/CO2 insufflation. METHODS: A 5-cm segment of rat colon was insufflated with either air (air group) or CO2 (CO2 group). Two insufflation methods were employed: temporary insufflation up to an intraluminal pressure of 60 mmHg and continuous insufflation at a pressure of 5, 15, and 30 mmHg. Bowel distention and parietal blood flow measured by laser Doppler imaging were evaluated. RESULTS: For temporary insufflation, bowel distention was prolonged in the air group, whereas it rapidly resolved in the CO2 group. Parietal blood flow decreased in both groups; however, it recovered within 5 min in the CO2 group. For continuous insufflation, under 5 mmHg insufflation, blood flow decreased in the air group, whereas it increased in the CO2 group. Blood flow decreased in both groups under 15 mmHg insufflation; however, it decreased less in the CO2 group. There was a reverse relationship between insufflation pressure and blood flow difference. Inhibition of nitric oxide synthase, ATP-sensitive K+ channel, or heme oxygenase was ineffective against a CO2-induced increase in blood flow. CONCLUSION: CO2 insufflation preserved parietal blood flow not only by rapid resolution of bowel distention but also by its potential vasodilative effect.

Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment.

Heme oxygenase-1 (HO-1) is believed to represent a key enzyme for the protection of cells against "stress." Its overexpression in different types of human cancers supports the notion that HO-1 provides a growth advantage and contributes to cellular resistance against chemotherapy and radiotherapy. Given the poor survival rates of patients with pancreatic cancer due to its aggressive growth behavior and its exceptional resistance to all known forms of anticancer treatment, we have investigated the expression of HO-1 in human pancreatic cancer cells growth behavior and prognosis. Expression of HO-1 was analyzed in human pancreatic cancer samples in comparison with normal pancreas by quantitative PCR, Western blot, and confocal microscopy. The influence of radiotherapy and chemotherapy on HO-1 expression in pancreatic cancer cell lines was evaluated. Furthermore, HO-1 expression was specifically suppressed by small interfering RNA transfection and subsequently the alterations of growth behavior and resistance to anticancer treatment were tested. Human pancreatic cancer showed a 6-fold and 3.5-fold HO-1 up-regulation in comparison to normal pancreas based on mRNA and protein level, respectively (P < 0.05). Cancer tissues revealed marked HO-1 immunoreactivity in tumor cells and in tumor associated immunocytes. Treatment of the pancreatic cancer cell lines with gemcitabine or radiation strongly induced HO-1 expression. Targeted knockdown of HO-1 expression led to pronounced growth inhibition of the pancreatic cancer cells and made tumor cells significantly more sensitive to radiotherapy and chemotherapy. Therefore, specific inhibition of HO-1 expression may be a new option in pancreatic cancer therapy and may be used as sensitizer to chemotherapy and radiotherapy.

Involvement of heme oxygenase as antioxidant defense in soybean nodules.

OBJECTIVE: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress. MATERIALS AND METHODS: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analyzed. RESULTS: Treatment with 200 microM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 microM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced. CONCLUSION: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.

Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes.

OBJECTIVE: Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of biliverdin (BV), carbon monoxide (CO), and ferrous iron. The inducible HO isoform, HO-1, confers protection against ischemia/reperfusion (I/R)-injury in the heart. We hypothesized that HO-1 and its catalytic by-products constitute an antihypertrophic signaling module in cardiac myocytes. METHODS AND RESULTS: The G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) (30 nmol/l) stimulated a robust hypertrophic response in cardiac myocytes isolated from 1- to 3-day-old Sprague-Dawley rats, with increases in cell surface area (planimetry), sarcomere assembly (confocal laser scanning microscopy), and prepro-atrial natriuretic peptide (ANP) mRNA expression. Adenoviral overexpression of HO-1, but not beta-galactosidase, significantly inhibited ET-1 induced cardiac myocyte hypertrophy. The antihypertrophic effects of HO-1 were mimicked by BV (10 micromol/l) and the CO-releasing molecule [Ru(CO)3Cl2]2 (10 micromol/l), strongly suggesting a critical involvement of BV and CO in the antihypertrophic effects of HO-1. Both BV and CO suppressed extracellular signal-regulated kinases (ERK1/ERK2) and p38 mitogen-activated protein kinase (MAPK) activation by ET-1 stimulation. Moreover, BV and CO inhibited the prohypertrophic calcineurin/NFAT pathway. This inhibition occurred upstream from calcineurin because BV and CO inhibited NFAT activation in response to ET-1 stimulation but not in response to adenoviral expression of a constitutively active calcineurin mutant. Upstream-inhibition of the calcineurin/NFAT pathway by CO occurred independent from cGMP and cGMP-dependent protein kinase type I (PKG I). CONCLUSIONS: Heme oxygenase-1 and its catalytic by-products, BV and CO, constitute a novel antihypertrophic signaling pathway in cardiac myocytes. Biliverdin and CO inhibition of MAPKs and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products may promote their antihypertrophic effects.

Glutamate-induced cell death of immortalized murine hippocampal neurons: neuroprotective activity of heme oxygenase-1, heat shock protein 70, and sodium selenite.

HT22 immortalized hippocampal neurons serve as a cellular model system to study oxidative stress, an imbalance of cellular redox homeostasis. Glutamate induces HT22 cell death by inhibiting the uptake of cystine into the cells via the cystine/glutamate transport system xc-, thus leading to reduced levels of glutathione. Here, we show that glutamate-induced cell death is attenuated in HT22 cells overexpressing heat shock protein 70 or heme oxygenase-1. Moreover, supplementing the culture medium with sodium selenite completely protected HT22 against oxidative glutamate toxicity. In contrast, neither heat shock protein 70 nor heme oxygenase-1 expression or increased concentrations of sodium selenite protected HT22 cells against serum withdrawal-induced cell death. These data indicate that glutamate-induced cell death differs substantially from that induced by growth factor deprivation.

Haem oxygenase augments porcine granulosa cell apoptosis in vitro.

Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 microM haemin (a haem oxygenase substrate) was approximately 1.7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity.

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-(S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 microM AA or 100 microM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.

Different effects of constitutive nitric oxide synthase and heme oxygenase on pulmonary or liver metastasis of colon cancer in mice.

It has recently been reported that not only endogenous nitric oxide (NO) but also carbon monoxide (CO) produced by heme oxygenase (HO) have many physiological functions. The objective of the present study was to determine whether endogenous NO or CO is involved in the experimental pulmonary or liver metastasis of colon cancer in mice. Intravenous or intrasplenic injection of colon 26 cells from a mouse colon adenocarcinoma cell line resulted in multiple pulmonary or liver metastases. NG-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase (NOS), or zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG), a competitive inhibitor of HO, was administered to the mice only on the day of tumor inoculation. We assessed the number of tumor cells 24 h later and the outcome of metastases of the target organ. In the pulmonary metastasis model, L-NAME increased both the number of tumor cells 24 h later and outcome of metastases 18 days later, but did not have a significant effect on liver metastasis. On the other hand, metastasis to the liver, but not that to the lung, increased following administration of ZnDPBG. These results suggest that the activities of NOS and HO could influence experimental metastasis in an organ-specific manner.

Role of cyclooxygenase and haemoxygenase products in nitric oxide-independent vasodilatation in the porcine ciliary artery.

PURPOSE: Vascular endothelial cell dysfunction has been noted in patients with normal pressure glaucoma. Although nitric oxide (NO) accounts for a large proportion of vasorelaxation in the posterior ciliary artery, considerable relaxation remains unexplained. We investigated the roles of haemoxygenase (HO) and cyclooxygenase (COX), which produce the vasodilators carbon monoxide (CO) and prostacyclin, respectively, in NO-independent endothelium-dependent vasodilatation in porcine posterior ciliary arteries. METHODS: Isolated vascular rings were mounted in a Mulvaney-Halpern small vessel myograph for the measurement of isometric tension development. Vasodilator responses to bradykinin (BK) were elicited in each ring on three separate occasions following preconstriction with prostaglandin F(2alpha): first in the absence of inhibitors, second in the presence of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 10(-3) M), and third in the presence of L-NAME and either a COX (indomethacin, 10(-6) M) or an HO inhibitor (tin protoporphyrin-IX 10(-5) M). Results were expressed as a percentage of the maximal relaxation in the presence of L-NAME alone. RESULTS: Incubation with indomethacin (n=6), in the presence of L-NAME, significantly reduced (P<0.01) maximum BK-induced relaxation (-103.5+/-8.8%) compared to paired rings in the presence of L-NAME alone (-130.8+/-8.8%). HO inhibition did not reduce NO-independent, BK-induced relaxation when compared to paired control vessels. CONCLUSIONS: These data suggest that in the presence of L-NAME, a COX product accounts for a significant proportion of NO-independent vasodilatation. In contrast, endogenous CO production does not have a functionally significant role in the porcine ciliary artery. Eye (2003) 17, 628-636. doi:10.1038/sj.eye.6700437

Prevention of chronic deterioration of heart allograft by recombinant adeno-associated virus-mediated heme oxygenase-1 gene transfer.

BACKGROUND: Allograft deterioration is the major obstacle to organ transplantation as a long-term treatment of end-stage heart failure. In this study, we transduced the antioxidant gene, heme oxygenase-1 (HO-1), to heart grafts using a recombinant adeno-associated viral vector (rAAV) in a rat heart transplantation model and investigated its potentiality in prevention of chronic graft deterioration. METHODS AND RESULTS: rAAV/HO-1 was administered to heart grafts through the coronary arteries during cold preservation. We investigated the expression patterns and activities of transgene, graft survival, graft histomorphology, and relevance of HO-1 expression on graft survival and chronic graft deterioration by itself. Long-term allograft survival can be achieved by rAAV/HO-1-mediated stable transgene expression. The development of graft arteriosclerosis and interstitial fibrosis was prevented in rAAV/HO-1-transduced allografts on day 100. rAAV/HO-1-mediated long-term graft protection was accompanied by remarkable downregulation of the intragraft mRNA level of macrophage migration inhibitory factor, tumor necrosis factor-alpha, and transforming growth factor-beta1. Blockage of HO activities by zinc protoporphyrin IX at different posttransplant phases showed that the stable expression of HO-1 is a prerequisite for both survival of grafts and prevention of graft arteriosclerosis. CONCLUSIONS: rAAV/HO-1 gene transfer represents a novel therapeutic approach to prevent chronic allograft deterioration in clinical heart transplantation.