Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.     

Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse

Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1(-/-)) and wild-type (HO-1(+/+)) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1(-/-) mice. The lymph nodes of HO-1-deficient mice demonstrated a relative paucity of CD3- and B220-positive cells, but no such abnormalities were observed in the thymus. Flow cytometric analysis of isolated splenocytes demonstrated no differences in the proportions of T lymphocytes, B lymphocytes or monocytes/macrophages between the HO-1(-/-) and HO-1(+/+) mice. Significantly higher baseline serum IgM levels were observed in HO-1(-/-) versus HO-1(+/+) mice. Under mitogen stimulation with either lipopolysaccharide or anti-CD3/anti-CD28, HO-1(-/-) splenocytes secreted disproportionately higher levels of pro-inflammatory Th1 cytokines as compared to those from HO-1(+/+) mice. These findings demonstrate significant differences in the immune phenotype between the HO-1(-/-) and the HO-1(+/+) mice. The absence of HO-1 correlates with a Th1-weighted shift in cytokine responses suggesting a general pro-inflammatory tendency associated with HO-1 deficiency.     

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.     

Heme oxygenase-1: unleashing the protective properties of heme

Heme oxygenase (HO)-1 catabolizes heme into three products: carbon monoxide (CO), biliverdin (which is rapidly converted to bilirubin) and free iron (which leads to the induction of ferritin, an iron-binding protein). HO-1 serves as a "protective" gene by virtue of the anti-inflammatory, anti-apoptotic and anti-proliferative actions of one or more of these three products. Administration of CO, biliverdin, bilirubin or iron-binding compounds is protective in rodent disease models of ischemia-reperfusion injury, allograft and xenograft survival, intimal hyperplasia following balloon injury or as seen in chronic graft rejection and others. We suggest that the products of HO-1 action could be valuable therapeutic agents and speculate that HO-1 functions as a "therapeutic funnel", mediating the beneficial effects attributed to other molecules, such as interleukin-10 (IL-10), inducible nitric oxide synthase (NOS2; iNOS) and prostaglandins. This Review is the third in a series on the regulation of the immune system by metabolic pathways.     

丹参对大鼠移植肝血红素氧合酶1表达的影响

背景：器官移植前使用丹参预处理能够保护组织缺血-再灌注损伤,改善移植器官存活率。 目的：观察含丹参的冷灌注液对同种异体大鼠移植肝脏中血红素氧合酶1表达的影响,以及对供体肝脏缺血-再灌注损伤的保护作用。 方法：将SD雄性大鼠随机分成UW液组(术中使用UW液灌注保存)、丹参+UW液组(术中使用丹参+UW液灌注保存)、ZnPP预处理组(移植前24 h腹腔内注射ZnPP,术中使用丹参+UW液灌注保存),建立稳定的大鼠同种异体肝移植模型。同时取10只正常大鼠作为正常对照。 结果与结论：丹参+UW液组和UW液组血清总胆红素、谷丙转氨酶、谷草转氨酶水平明显低于ZnPP预处理组(P < 0.01)。血红素氧合酶1mRNA及其蛋白在丹参+UW液组中较UW组表达更明显,在ZnPP预处理组中表达明显受到抑制(P< 0.05)。丹参+UW液组肝脏Suzuki标准评分明显低于ZnPP预处理组及UW液组(P < 0.05)。表明丹参能上调同种异体的大鼠移植肝脏中血红素氧合酶1 mRNA及其蛋白的表达,减轻供肝缺血-再灌注损伤,保护移植大鼠肝脏。     

高浓度氧增加未成年大鼠肺血红素加氧酶-1表达

目的 探讨高浓度氧对未成年大鼠肺血红素加氧酶-1(HO-1)表达的影响.方法 将生后21 d SD大鼠40只随机分为空气组和12、24、48及72 h高氧组,分别置于空气和常压高氧箱(92%~94% O<,2>)中.检测左肺湿/干重比及肺组织病理学改变,用RT-PCR和Western blot法分别检测肺HO-1 mRNA及HO-1蛋白表达情况.结果 高氧12 h组肺HO-1 mRNA吸光度积分相对值和高氧24 h组HO-1蛋白表达水平分别为0.350±0.043和0.455±0.046,较对照组0.263±0.037和0.280±0.044明显升高,且均随高氧暴露时间延长而表达进一步增加(P<0.05,P<0.01).与空气组比较,高氧48 h组和高氧72 h组左肺湿重/干重及肺损伤评分显著增高(P<0.05,P<0.01).结论 高浓度氧可引起未成年大鼠肺组织HO-1表达增多.     

Expression of heme oxygenase-1 in rat ontogeny

Heme oxygenase (HO), the heme-degrading enzyme, plays an important role in heme catabolism. Among three isozymes, HO-1 is an inducible form expressed mainly in macrophages. In rat ontogeny, HO-1 immunoreactivity was detected in mononuclear cells in the yolk sac at 10 days of gestation. HO-1-expressing cells were then detected in the fetal liver and their numbers increased during the gestational period. The numbers of HO-1-positive cells and HO-1 mRNA levels in the liver peaked at 18 days of gestation. Most of the macrophages expressed both HO-1 and a macrophage scavenger receptor. Macrophages in the fetal liver showed marked hemophagocytosis. Macrophages in the lung, spleen, bone marrow, and other tissues also expressed HO-1. HO-1 immunoreactivity was also observed in syncytial cells of the chorionic villi, the endodermal layer of the yolk sac, and renal tubules of the fetus. Intestinal mucosal epithelial cells expressed HO-1 after birth. These findings imply that HO-1 is crucial for macrophages in heme catabolism from an early stage of ontogeny. HO-1 expression in non-macrophagic cells may be required for other purposes such as protection from oxidative stress and various stimuli.     

Heme oxygenase-1 modulates fetal growth in the rat

Intrauterine growth restriction is associated with increased perinatal morbidity and mortality as well as with lifelong cardiovascular and metabolic complications. Deficiency of heme oxygenase 1 (HO-1) is associated with growth restriction in mice and in humans, suggesting a role for HO-1 in fetal growth and maintenance of pregnancy. We hypothesized that modulation of HO-1 in the pregnant rat would alter fetal growth. In pregnant dams, placental HO activity was significantly inhibited with zinc deuteroporphyrin IX 2,4 bis glycol, and HO-1 protein was increased by transducing adenoviral human HO-1. Inhibition of HO-1 by zinc deuteroporphyrin IX 2,4 bis glycol resulted in a significant decrease in pup size, whereas transfection with hHO-1 resulted in increased pup size. Furthermore, the expression of IGF binding protein-1 and its receptor paralleled the expression of HO-1 in the placenta and were significantly modulated by modification of HO-1 along with the expression of vascular endothelial growth factor. These observations demonstrate that HO-1 modulates fetal growth by its effects on placental growth factors.     

Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs

Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys264-Pro265 and Cys281-Pro282. We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP nor PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each HRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys264-Pro265 result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.     

Isoliquiritigenin, from Dalbergia odorifera, up-regulates anti-inflammatory heme oxygenase-1 expression in RAW264.7 macrophages

Objectives:  Isoliquiritigenin (ISL), one of the major constituents of Dalbergia odorifera T. Chen (Leguminosae), is reported to exert anti-inflammatory effects, but the relevant anti-inflammatory mechanisms are not completely understood. Heme oxygenase-1 (HO-1) has been proven to be involved in the resolution of inflammatory responses. In this study, we investigated whether ISL could induce HO-1 expression in RAW264.7 macrophages, and if so, whether HO-1 could mediate the anti-inflammatory effects of ISL. Methods:  The protein expression of inducible nitric oxide synthase and HO-1 was analyzed by western blot analysis. The production of nitric oxide (NO) and interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was assayed by Griess and ELISA, respectively. The TNF-α and HO-1 mRNA expression was analyzed by northern blot analysis. Results:  ISL markedly suppressed LPS-induced NO, IL-1β, and TNF-α production. ISL induced HO-1 expression through the extracellular signal-regulated kinase1/2 pathway in RAW264.7 macrophages. The effects of ISL on LPS-induced NO and TNF-α production were reversed by the HO-1 inhibitor, tin protoporphyrin. Conclusions:  ISL is an effective HO-1 inducer capable of inhibiting macrophage-derived inflammation. Received 13 August 2008; returned for revision 15 September 2008; returned for final revision 28 October 2008; accepted by G. Wallace 24 November 2008     

Heme oxygenase-1 protects brain from acute excitotoxicity

Heme oxygenase is a rate-limiting enzyme that degrades heme, a pro-oxidant, into carbon monoxide, iron, and bilirubin. Heme oxygenase has two active isoforms: heme oxygenase-1 and heme oxygenase-2. Heme oxygenase-1 can be induced by various insults. Several investigators have postulated that it has cytoprotective activities, although its role in the nervous system is not fully understood, especially considering that normally heme oxygenase-2 accounts for the vast majority of heme oxygenase activity in the brain. Here, the basal effect of heme oxygenase-1 was investigated in acute glutamatergic excitotoxicity to test the hypothesis that N-methyl-D-aspartate-induced acute toxicity in brain is attenuated by heme oxygenase-1. N-methyl-D-aspartate was unilaterally injected into the striatum of wildtype and heme oxygenase-1 knockout mice. After 48 h, brains were harvested, sectioned, and stained with Cresyl Violet to measure the lesion size. Lesion volume was significantly (P<0.05) greater in brains of heme oxygenase-1 knockout mice (15.2+/-3.1 mm(3); n=10) than in those of wildtype mice (6.2+/-1.5 mm(3); n=11). In addition, Western blot analysis indicated no detectable differences between wildtype and heme oxygenase-1 knockout mouse brains in the levels of the glutamate or N-methyl-D-aspartate receptors studied. To test whether heme oxygenase-1 could specifically protect neurons, mouse primary neuronal cell cultures of wildtype and heme oxygenase-1 knockout mice were treated with or without N-methyl-D-aspartate. Cell viability of the heme oxygenase-1 knockout neurons was significantly less than that of wildtype neurons at each of the N-methyl-D-aspartate concentrations tested (12.8+/-1.3%, 16.0+/-1.4%, and 18.4+/-1.8% at 30, 100, and 300 microM N-methyl-D-aspartate, respectively). These results indicate that heme oxygenase-1 provides neuroprotection against acute excitotoxicity and suggest that potential intervention that can increase heme oxygenase-1 activity within the brain should be considered as a therapeutic target in acute and potentially chronic neurological disorders.     

Heme oxygenase-1 and chronic hypoxia

A myriad of changes are necessary to adapt to chronic hypoxemia. Key among these changes increases in arterial oxygen carrying capacity, ventilation and sympathetic activity. This requires the induction of several gene products many of which are regulated by the activity of HIF-1α, including HO-1. Induction of HO-1 during chronic hypoxia is necessary for the continued breakdown of heme for the enhanced production of hemoglobin and the increased respiratory and sympathetic responses. Several human HO-1 polymorphisms have been identified that can affect the expression or activity of HO-1. Associations between these polymorphisms and the prevalence of hypertension have recently been assessed in specific populations. There are major gaps in our understanding of the mechanisms of how HO-1 mediates changes in the activity of the hypoxia-sensitive chemosensors and whether HO-1 polymorphisms are an important factor in the integrated response to chronic hypoxia. Understanding how HO-1 mediates cardiorespiratory responses could provide important insights into clinical syndromes such as obstructive sleep apnea.     

Heme oxygenase-1 and transplantation tolerance

Tolerance is the ultimate goal of transplantation: the lack of any need for immunosuppression with long-term survival of the transplanted tissue and a full immunological response to all antigens other than those on the transplant. In honor of my dear friend and colleague, Prof. Dr. Jon van Rood, I summarize here our work in this area making use of heme oxygenase-1 (HO-1) and the products that degradation of heme by HO-1 generates.     

Induction of heme oxygenase-1 expression inhibits platelet-dependent thrombosis

Heme oxygenase-1 (HO-1) plays a key role in protecting tissue from oxidative stress. Although some studies implicate HO-1 in modulating thrombosis after vascular injury, the impact of HO-1 on the rate of clot formation in vivo is poorly defined. This study examined the potential function of HO-1 in regulating platelet-dependent arterial thrombosis. Platelet-rich thrombi were induced in C57BL/6J mice by applying 10% ferric chloride to the exposed carotid artery. Mean occlusion time of wild-type mice (n = 10) was 14.6 +/- 1.0 min versus 12.9 +/- 0.6 min for HO-1-/- mice (n = 11, p = 0.17). However, after challenge with hemin, mean occlusion time was significantly longer in wild-type mice (16.3 +/- 1.2 min, n = 15) than HO-1-/- mice (12.0 +/- 1.0 min, n = 9; p = 0.021). Hemin administration induced an approximately twofold increase in oxidative stress, measured as plasma thiobarbituric acid reactive substances. Immunohistochemical analysis revealed that hemin induced a robust increase in HO-1 expression within the carotid arterial wall. Ex vivo blood clotting within a collagen-coated perfusion chamber was studied to determine whether the accelerated thrombosis observed in HO-1-/- mice was contributed to by effects on the blood itself. Under basal conditions, mean clot formation during perfusion of blood over collagen did not differ between wild-type mice and HO-1-/- mice. However, after hemin challenge, mean clot formation was significantly increased in HO-1-/- mice compared with wild-type controls. These results suggest that, under basal conditions, HO-1 does not exert a significant effect on platelet-dependent clot formation in vivo. However, under conditions that stimulate HO-1 production, platelet-dependent thrombus formation is inhibited by HO-1. Enhanced HO-1 expression in response to oxidative stress may represent an adaptive response mechanism to down-regulate platelet activation under prothrombotic conditions.     

诱导HO-1基因表达的信号通路

血红素氧合酶（HO）的诱导型HO-1与其催化血红素降解生成的产物胆红素和CO一道，组成了机体重要的内源性保护系统，广泛参与抗炎与多种急慢性氧化应激损伤。多种理化因素通过不同的细胞内信号转导通路诱导HO-1的表达，这些信号通路涉及丝裂原活化蛋白激酶（MAPKs）、蛋白激酶C(PKC)、cAMP依赖的蛋白激酶A(PKA)、cGMP依赖蛋白激酶G（PKG）、酪氨酸蛋白激酶（TPK）、蛋白磷酸酶（PPs）、磷脂酰肌醇（-3）激酶（PI3K）/Akt     

Glial heme oxygenase-1 expression in Alzheimer disease and mild cognitive impairment

We determined whether oxidative stress is an early event in the pathogenesis of sporadic Alzheimer disease (AD), and correlated oxidative stress with neuropsychological functions and neurofibrillary pathology in AD and mild cognitive impairment (MCI). Oxidative stress was measured as the percentage of astrocytes expressing heme oxygenase-1 (HO-1) in post mortem temporal cortex and hippocampus after dual HO-1/glial fibrillary acidic protein (GFAP) immunohistochemistry. Glial HO-1 expression in the MCI temporal cortex and hippocampus was significantly greater than in the non-demented group and did not differ from AD values. Astroglial HO-1 expression in the temporal cortex was associated with decreased scores for global cognition, episodic memory, semantic memory and working memory. Hippocampal astroglial HO-1 expression was associated with lower scores for global cognition, semantic memory and perceptual speed. Glial HO-1 immunoreactivity in the temporal cortex, but not hippocampus, correlated with the burden of neurofibrillary pathology. Cortical and hippocampal oxidative stress is a very early event in the pathogenesis of sporadic AD and correlates with the development of specific cognitive deficits in this condition.