Heme Oxygenase Type Research Articles

A Review of Oxidative Stress Products and Related Genes in Early Alzheimer's Disease.

Oxidative stress is associated with the progression of Alzheimer’s disease (AD). Reactive oxygen species can modify lipids, DNA, RNA, and proteins in the brain. The products of their peroxidation and oxidation are readily detectable at incipient stages of disease. Based on these oxidation products, various biomarker-based strategies have been developed to identify oxidative stress levels in AD. Known oxidative stress-related biomarkers include lipid peroxidation products F2-isoprostanes, as well as malondialdehyde and 4-hydroxynonenal which both conjugate to specific amino acids to modify proteins, and DNA or RNA oxidation products 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG), respectively. The inducible enzyme heme oxygenase type 1 (HO-1) is found to be upregulated in response to oxidative stress-related events in the AD brain. While these global biomarkers for oxidative stress are associated with early-stage AD, they generally poorly differentiate from other neurodegenerative disorders that also coincide with oxidative stress. Redox proteomics approaches provided specificity of oxidative stress-associated biomarkers to AD pathology by the identification of oxidatively damaged pathology-specific proteins. In this review, we discuss the potential combined diagnostic value of these reported biomarkers in the context of AD and discuss eight oxidative stress-related mRNA biomarkers in AD that we newly identified using a transcriptomics approach. We review these genes in the context of their reported involvement in oxidative stress regulation and specificity for AD. Further research is warranted to establish the protein levels and their functionalities as well as the molecular mechanisms by which these potential biomarkers are involved in regulation of oxidative stress levels and their potential for determination of oxidative stress and disease status of AD patients.

Cytotoxic effect of hemin in colonic epithelial cell line: Involvement of 18 kDa translocator protein (TSPO)

AimsThe aim of this study is to investigate the effect of hemin in colonic epithelial cells (Caco-2) cell proliferation and if this effect was due to a direct modulation of 18-kDa translocator protein (TSPO) and/or heme oxygenase type 1 (HO-1). Main methodsThe main methods are as follow: cell proliferation and cell cytotoxic assays on Caco-2 cell lines treated with hemin in the presence or not of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) and Sn-protoporphyrin IX (Sn-PPIX), and immunoblotting for TSPO and HO-1 protein analysis, siRNA directed against TSPO. Key findingsHemin was shown to be toxic for the Caco-2 cell line in a concentration and time dependent manner. Although hemin was able to induce HO-1 in a dose dependent manner, a specific HO-1 inhibitor, Sn-PPIX, was unable to interfere with the effect of hemin on Caco-2 cells. Instead, PK 11195, a specific TSPO ligand, was able to counteract the effect of hemin, suggesting an important role of TSPO in the hemin activity. Cell viability assay further confirms the high cytotoxic effects exerted by hemin on Caco-2 cells expressing TSPO compared to the siRNA-TSPO targeted cells. In addition, hemin was able to decrease significantly the TSPO protein density in a dose dependent manner after 24h of incubation. SignificanceThe interaction and the consecutive down regulation of TSPO by hemin played an important role in the control of Caco-2 cell viability. The presented data suggest that TSPO might contribute to protect cells from potential toxic compounds such as free tetrapyrroles, candidating this receptor as a survival receptor protein.

Diphenyl diselenide administration enhances cortical mitochondrial number and activity by increasing hemeoxygenase type 1 content in a methylmercury-induced neurotoxicity mouse model

Interest in biochemistry of organoselenium compound has increased in the last decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of diphenyl diselenide (PhSe)2 (5 μmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Swiss male mice were divided into four experimental groups: control, (PhSe)2 (5 μmol/kg, subcutaneous administration), MeHg (40 mg/L, in tap water), and MeHg + (PhSe)2. After the treatment (21 days), the animals were killed and the cerebral cortex was analyzed. Electron microscopy indicated an enlarged and fused mitochondria leading to a reduced number of organelles, in the MeHg-exposed mice. Furthermore, cortical creatine kinase activity, a sensitive mitochondrial oxidative stress sensor, was almost abolished by MeHg. Subcutaneous (PhSe)2 co-treatment rescued from MeHg-induced mitochondrial alterations. (PhSe)2 also behaved as an enhancer of mitochondrial biogenesis, by increasing cortical mitochondria content in mouse-receiving (PhSe)2 alone. Mechanistically, (PhSe)2 (1 μM; 24 h) would trigger the cytoprotective Nrf-2 pathway for activating target genes, since astroglial cells exposed to the chalcogen showed increased content of hemeoxygenase type 1, a sensitive marker of the activation of this via. Thus, it is proposed that the (PhSe)2-neuroprotective effect might be linked to its mitoprotective activity.

Carbon monoxide stimulates Ca2+-dependent big-conductance K channels in the cortical collecting duct

We used the patch-clamp technique to examine the role of carbon monoxide (CO) in regulating Ca(2+)-activated big-conductance K (BK) channels in the principal cell of the cortical collecting duct (CCD). Application of CORM3 or CORM2, a CO donor, activated BK channels in the CCD, whereas adding inactivated CORM2/3 had no effect. Superfusion of the CCD with CO-bubbled bath solution also activated the BK channels in the cell-attached patches. The effect of CO on BK channels was not dependent on nitric oxide synthase (NOS) because the effect of CORM3 was also observed in the CCD treated with l-NAME, an agent that inhibits the NOS. Adding a membrane-permeable cGMP analog, 8-bromo-cGMP, significantly increased the BK channel in the CCD. However, inhibition of soluble guanylate cyclase failed to abolish the stimulatory effect of CORM3 on BK channels. Moreover, inhibition of cGMP-dependent protein kinase G did not block the stimulatory effect of CORM3 on the BK channels, suggesting that the stimulatory effect of CO on the BK channels was, at least partially, induced by a cGMP-independent mechanism. Western blot demonstrated that heme oxygenase type 1 (HO-1) and HO-2 were expressed in the kidney. Moreover, a high-K (HK) intake increased the expression of HO-1 but not HO-2 in the kidney. A HK intake also increased renal HO activity defined by NADPH-dependent CO generation following addition of heme in the cell lysate from renal cortex and outer medulla. The role of HO in regulating BK channel activity in the CCD was also suggested by experiments in which application of hemin increased the BK channels. The stimulatory effect of hemin on the BK channels was blocked by SnMP, a HO inhibitor. But, adding CORM3 was still able to activate the BK channels in the presence of SnMP. We conclude that CO activates the BK channels, at least partially, through a NO-cGMP-independent pathway and that HO plays a role in mediating the effect of HK intake on the BK channels in the CCD.

Enhanced Expression of Heme Oxygenase‐1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 μm) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Hypoxia-Mediated Induction of Heme Oxygenase Type I and Carbon Monoxide Release from Astrocytes Protects Nearby Cerebral Neurons from Hypoxia-Mediated Apoptosis

To study a putative paracellular protective mechanism of astrocytes for neurons, immunohistochemical analysis was performed in ischemic rat brain, which colocalized with the expression of heme oxygase-1 (HO- 1) in astroglias surrounding dying TUNEL-positive neurons. As an in vitro paradigm for ischemia, cultured astrocytes were exposed to normobaric hypoxia (pO(2) asymptotically equal to 10 torr), which triggered marked increase in the expression of a 33 kDa stress protein, identified as HO-1. Induction of HO-1 message was observed within 4 h of hypoxia and peaked at 12 h, accompanied by an accelerated transcription of HO-1 message. Consistent with the induction of HO-1, a platelet bioassay revealed production of carbon monoxide by reoxygenated astrocytes. The presence of CO in the medium decelerated the hypoxia-mediated apoptotic type of cell death in cultured cerebral neurons via lowering the activity of caspase-3, a key enzyme regulating apoptotic cell death. This protection against apoptosis was likely mediated by CO-mediated increases in intracellular cGMP, because exposure of hypoxic neurons to CO increased intracellular cGMP levels, and addition of cGMP analogue to hypoxic neuronal cultures suppressed caspase-3 activity and promoted neuronal survival. These data describe a potentially important paracellular pathway through which astrocytes may rescue nearby neurons from ischemic death.

Formalin-induced spinal cord calcium/calmodulin-dependent protein kinase IIα expression is modulated by heme oxygenase in mice

The injection of formalin into the hindpaws of rats and mice is widely used as a model of inflammatory pain. The allodynia observed in this model is due in part to sensitization of spinal cord dorsal horn neurons, a form of neuroplasticity similar to long-term potentiation in the hippocampus. Ca 2+/calmodulin-dependent kinase type IIα (CaMKIIα) is a key component of long-term potentiation. Here we report alterations in CaMKIIα mRNA and protein expression in spinal cord tissue from wild-type and heme oxygenase type 2 (HO-2) null mutant mice after formalin injection. Behavioral experiments demonstrated a long lived allodynia in wild-type C57Bl/6J mice after hindpaw formalin injection, but less in null mutant mice. Both CaMKIIα mRNA and protein expression were increased in a time-dependent manner in the spinal cords of wild-type mice after formalin injection. Confocal microscopy localized the increased expression to the superficial laminae of the spinal cord dorsal horn. In the HO-2 null mutant mice no significant change in CaMKIIα mRNA expression and only a small increase in protein were noted. These findings suggest that time-dependent CaMKIIα expression may underlie central sensitization and allodynia induced by hindpaw formalin injection, and that this process is modulated by HO-2.

Heme oxygenase type 2 modulates behavioral and molecular changes during chronic exposure to morphine

The heme oxygenase (HO) enzyme system has been shown to participate in nociceptive signaling in a number of different models of pain. In these experiments we investigated the role of the HO type 2 (HO-2) isozyme in tolerance to the analgesic effects of morphine, and the hyperalgesia and allodynia which are measurable upon cessation of administration. Wild type C57Bl/6 wild type mice or HO-2 null mutants in that background strain were treated with morphine for 5 days. The morphine administration protocol consisted of either twice daily repeated s.c. boluses of 15 mg/kg or s.c. implantation of a morphine pellet. At the end of the treatment period wild type mice treated by either protocol exhibited tolerance, but the HO-2 null mutants did not. The HO-2 null mutants also exhibited less mechanical allodynia following cessation of morphine administration, though only modest differences in thermal hyperalgesia were noted. There was no correlation between the degree of tolerance obtained in the bolus and pellet protocols and the degree of hyperalgesia and allodynia observed after cessation of morphine administration in the wild type mice. Our final experiments analyzed increases in expression of mRNA for nitric oxide synthase type 1, N-methyl- d-aspartate (NMDA) receptor NMDAR1 subunit and prodynorphin in spinal cord tissue. In pellet-treated mice two- to three-fold increases were observed in the abundance of these species, but very little change was observed in the null-mutant mice. Taken together our results indicate that HO-2 participates in the acquisition of opioid tolerance, the expression of mechanical allodynia after cessation of opioid administration and in gene regulation occurring in the setting of treatment with morphine. Furthermore, these studies suggest that the mechanisms underlying analgesic tolerance and opioid-induced hypersensitivity are at least somewhat distinct.

Heme oxygenase type 2 participates in the development of chronic inflammatory and neuropathic pain

Abstract: Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, iron, and carbon monoxide. Recent studies have demonstrated that inhibitors of HO are analgesic in a number of different pain models. In these studies we attempted to define the role of HO type 2 (HO-2) in the development of chronic inflammatory and neuropathic pain. To do this both wild type and C57Bl/6 HO-2 null mutant mice were either injected with complete Freund[apos ]s adjuvant in 1 hind paw or underwent unilateral partial sciatic nerve ligation. The resulting thermal hyperalgesia and mechanical allodynia were monitored for up to 14 days afterward. In both models of chronic pain it was observed that the extent of hyperalgesia and allodynia was significantly less for the HO-2 null mutants than the wild type mice. Additional studies quantified spinal cord dorsal horn Fos expression after brushing of the affected hind paw for both complete Freund[apos ]s adjuvant and partial sciatic nerve ligation treated mice. These studies showed that HO-2 null mutants had less Fos expression after stimulation by brushing than did their wild type counterparts. Our results indicate that HO-2 participates in the thermal hyperalgesia and mechanical allodynia that occur in 2 commonly used models of chronic inflammatory and neuropathic pain. [copy ] 2003 by the American Pain Society

Spinal cord heme oxygenase participates in glutamate-induced pain-related behaviors

Heme oxygenase catalyzes the formation of CO, Fe 2+ and biliverdin from the substrate heme. In these studies, we attempted to define the roles heme oxygenase play in pain-related behaviors induced by intrathecal injection of the spinal neurotransmitter glutamate. The intrathecal injection of glutamate or the more selective agonists N-methyl- d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in C57Bl/6 mice lead to caudally directed pain behaviors which were sensitive to the heme oxygenase inhibitors tin protoporphyrin (Sn-protoporphyrin) and chromium mesoporphyrin (Cr-mesoporphyrin). Intrathecal injections of glutamate in heme oxygenase type 2 (HO-2) null-mutant animals resulted in reduced pain-related behaviors when compared with wild type animals. Glutamate, NMDA and AMPA stimulated cGMP accumulation in mouse spinal cord slices, which was blocked by heme oxygenase inhibitors. Glutamate did not stimulate cGMP production in HO-2 null-mutant animals. Our data are consistent with the hypothesis that pain-related behaviors induced by spinal glutamate rely on the activation of HO-2 and subsequent production of cGMP.

Gene Expression of Nitric Oxide Synthase and Heme Oxygenase in Placental Villi during Pregnancy with and without Intrauterine Growth Restriction

It is commonly accepted that the intrauterine growth restriction (IUGR) is correlated with an impairment of utero-placental blood flow. This blood flow is regulated by prostaglandins and nitric oxide (NO), which regulate vascular tone in placental villi. The factors that regulate utero-placental blood flow play different functions independently, and their functions differ according to the gestational stage. To evaluate the influence of NO and carbon monoxide (CO) on the feto-maternal circulation, we quantified endothelial NO synthase (eNOS) and heme oxygenase (HO) type 1 (HO-1) mRNAs in the placental villi. Forty-three placental samples were employed. We measured both eNOS and HO-1 mRNAs by Northern blot hybridization. The eNOS mRNA expression in the first trimester (relative intensity, 3.741±0.679, mean±SEM) was higher than that after the first trimester (0.500±0.038, p<0.0001). The HO-1 mRNA expression in the third trimester was higher than that before the third trimester (2.648±0.409 and 1.122±0.182, respectively, p<0.005). There was no difference in the expression of eNOS and HO-1 mRNAs between pregnancies with or without IUGR. Independent expression of eNOS and HO-1 mRNAs suggests that eNOS is important in placentation at early gestation, whereas HO-1 is important in maintenance of pregnancy for the term.

Heme oxygenase inhibitors reduce formalin-induced Fos expression in mouse spinal cord tissue

Recent work from our laboratory and others supports a role for heme oxygenase in nociception and pain of several etiologies including inflammatory, incisional and neuropathic. Since it has been observed that heme oxygenase inhibitors reduce formalin-induced pain behaviors in mice and rats, we attempted to determine if this analgesic effect was reflected in a reduction in formalin-induced spinal cord Fos expression, an index of neuronal activation. To perform these studies, it was necessary to first examine the cytoarchitecture of the mouse lumbar spinal cord so that histological sections from known segmental levels could be chosen, and Fos-positive nuclei could be assigned to established dorsal horn laminae. After documenting the segmental and laminar distribution of Fos-positive nuclei following a 5% formalin injection, we went on to determine that the heme oxygenase inhibitor tin-protoporphyrin or morphine reduced this Fos expression as analyzed using confocal fluorescence microscopy. It was also observed that mice lacking expression of heme oxygenase type 2, an isozyme of heme oxygenase found in high abundance in the spinal cord, had lowered Fos expression after the formalin injection. Additional confocal microscopy studies demonstrated widespread expression of heme oxygenase type 2 in spinal cord neuron cell bodies. Double-labeling experiments showed that a high percentage of Fos-positive nuclei identified after administration of formalin were located within heme oxygenase type 2-positive cell profiles. Our studies support the hypothesis that heme oxygenase type 2 plays a role in formalin-induced nociception. Furthermore, from these results we suggest that the heme oxygenase type 2 located in spinal cord dorsal horn neurons participates in this nociceptive pathway.

Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

Heme oxygenase type 2 plays a role in formalin-induced nociception

Although much attention has been focused in recent years on nitric oxide synthase (NOS) as an enzyme intimately involved in many types of nociceptive signaling, the enzyme heme oxygenase (HO) has received little attention. Yet, HO produces gaseous second messenger molecule CO which, like NO, has proven to be an important neurotransmitter in the CNS. In these studies we provide detailed evidence that HO activity is critical to formalin-induced licking behavior in mice. The HO inhibitor tin protoporphyrin (Sn-P) dose-dependently reduced formalin-stimulated licking behavior in both phases of the formalin assay. This apparent analgesic effect was unlikely due to the non-specific effects of this agent as Sn-P did not alter rotarod performance, and the blood–brain barrier impermeant HO inhibitor zinc protoporphyrin (Zn-P) had little effect on licking times. We also hypothesized that heme oxygenase type 2 (HO-2) was the specific isoform of HO involved in nociception. Mice with a targeted disruption of the HO-2 gene were found to have greatly reduced licking times. Furthermore, Sn-P did not further reduce licking times when administered to HO-2 knockout animals. Taken together our evidence indicates that HO plays an important role in nociceptive signaling related to inflammatory-type pain, and that HO-2 is the isozyme mediating this nociception.

Chronic morphine exposure and the expression of heme oxygenase type 2

Heme oxygenase (HO) catalyzes the formation of carbon monoxide (CO) and other products from heme. The CO formed has been shown to function as a neurotransmitter, and may be involved in nociceptive signaling. Heme oxygenase type 2 (HO-2) is the predominant form of HO in the CNS. The expression of nitric oxide synthase (NOS) which catalyzes the formation of a similar neurotransmitter nitric oxide (NO) from arginine is increased in the spinal cords of animals chronically exposed to morphine and other opioids. In these studies, we examined changes in expression of HO-2 which occur in spinal cord tissue of morphine tolerant mice. After 5 days of exposure to morphine, mice were observed to be profoundly tolerant to the analgesic effects of morphine. In experiments using Northern blotting we observed a 2.7-fold increase in HO-2 mRNA in homogenized spinal cord tissue. Additional experiments revealed a 3.1-fold increase in HO-2 protein which seemed to result from the increased expression of HO-2 in neurons in the dorsal horn region of the spinal cord. To complement our expression studies measured HO enzymatic activity in spinal cord homogenates and found a 2.1-fold increase in the tolerant animals. The functional significance of this increased expression and activity is as yet unclear, but may be involved in the acquisition of analgesic tolerance to opioids, dependence on opioids, or perhaps the hyperalgesia reported after chronic exposure to opioids.

Induction of heme oxygenase type 1 (HO-1) in astrocytes by hypoxia and release of carbon monoxide (CO), a gas transmitter which suppresses a hypoxia-induced apoptotic type of cell death in cultured cerebellar neurons

Ich-1/Nedd2 and CPP32/YAMA are cysteine proteases related to interleukin 1-β-converting enzyme (ICE), which act as apoptosis effectors. Both molecules are expressed in T- and B-cell lines. The authors investigated their in vivo cellular distribution in normal and neoplastic human lymphoid tissues. Sixty-eight representative non-Hodgkin's lymphomas (NHL) and Hodgkin's disease (HD) samples, normal lymphoid organs, and nonlymphoid tumors were analyzed by immunohistochemistry (IHC). CPP32 expression in benign tissues was restricted to germinal center B cells, plasma cells, and a few interfollicular immunoblasts. All follicular NHLs and most diffuse large cell NHLs were CPP32 positive. Among T-cell NHLs, CPP32 expression was mainly observed in anaplastic large cell NHLs, whereas the other subtypes were less frequently positive. In contrast, lymphoid organs displayed only weak Ichl-L expression, located in sinusal histiocytes and thymic epithelial cells. Lymphomas were Ich1L negative, except for T-cell-rich B-cell NHLs, and about half of the HD samples, in which Reed-Sternberg cells (RSC) were usually Ich1L positive/CPP32 negative. Extralymphoid Ichl-L reactivity was found in particular organs like the kidney and various tumors. Western blot analysis confirmed the specificity of immunostaining. Neither CPP32 nor Ichl-L expression were correlated with intratumoral DNA fragmentation, as determined by the TUNEL assay. Altogether, these results indicate that CPP32 is preferentially expressed in germinal centers and thus could be involved in B-cell maturation. The differential expression of CPP32 and Ich1-L suggests that cysteine proteases differ in substrate specificities and carry out functions unrelated to apoptosis.

RAGE: a novel cellular receptor for advanced glycation end products.

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.