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.     

Cultured astrocytes from heme oxygenase-1 knockout mice are more vulnerable to heme-mediated oxidative injury

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO-2, the isoform expressed predominantly in neurons, accelerates heme-mediated neuronal injury, inhibitor studies suggest that HO-1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO-1 knockout and wild-type astrocytes to hemin. Consistent with prior observations, exposure of wild-type cultures to hemin for 24 hr resulted in protein carbonylation and concentration-dependent cell death between 10 and 60 microM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO-1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO-1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO-1 gene prior to hemin exposure. HO-2 expression was observed in both knockout and wild-type cultures and was not altered by HO-1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7-fold higher in knockout cells. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries.     

Induction of inducible heme oxygenase (HO-1) in the central nervous system: is HO-1 helpful or harmful?

Heme oxygenase (HO) produced biliverdin and bilirubin, which are powerful antioxidants, therefore, it has been proposed as helpful against oxidative stress. In contrast, HO also produces iron, and it could increase oxidative stress if not handled properly. To clarify the effect of HO, i.e., helpful or harmful, we examined the expression, localization, and induction mechanism of HO-1 in the rat hippocampus after transient forebrain ischemia and injection of kainic acid (KA). Following ischemia, HO-1 expression was observed early but transiently in the CA1 pyramidal neurons and later but continuously in glial cells. In addition, HO-1 expressing pyramidal neurons were colocalized well with phosphorylated c-Jun, which is a critical step in neuronal apoptosis. After injection of KA, HO-1 expression was observed only in glial cells but not neurons, and HO-1 expression was observed in predominantly ameboid microglia, along with a few astrocytes. HO-1 expressing ameboid microglia expressed major histocompatibility complex class II antigen, suggesting strong activation. These results suggested that HO-1 may have double-edged effects, and its effects may depend on the cell type. This short review is intended to highlight on the effect of HO-1 in neurodegeneration.     

Heme oxygenase-1 activity after excitotoxic injury: immunohistochemical localization of bilirubin in neurons and astrocytes and deleterious effects of heme oxygenase inhibition on neuronal survival after kainate treatment

An increased expression of the inducible form of heme oxygenase (HO), HO-1, is found in the hippocampus after kainate injection, but thus far it is unclear whether the HO-1 is enzymatically active. The present study was carried out, using monoclonal antibodies to bilirubin and HO-1 and histochemical staining for iron, to compare the products of HO enzymatic activity, bilirubin and iron, with HO-1 expression in the kainate-lesioned hippocampus. There was a close correlation between bilirubin and HO-1 expression, and both bilirubin and HO-1 were observed in damaged neurons at early times, and astrocytes at later times (weeks), after kainate injection. These results indicate that the increased HO-1 in the hippocampus is enzymatically active. Too determine whether HO-1 activity after kainate could have a protective or, perhaps, destructive effect, kainate-injected rats were injected intraperitoneally with a blood-brain barrier-permeable inhibitor of HO, tin protoporphyrin (SnPP), and the effects of such treatment were compared with effects in rats that received kainate and saline injection. It was found that SnPP treatment did not improve neuronal survival. Instead, increased mortality was observed in rats treated with SnPP. Four SnPP-injected rats vs. one saline-injected rats died after kainate treatment. The surviving SnPP-treated rats showed significantly less hippocampal field that containing Nissl or MAP2 staining (an indicator of surviving neurons) compared with the saline-injected rats. These results indicate that HO-1 induction had a net protective effect on neurons in the kainate model of excitotoxic injury.     

Hemoglobin neurotoxicity is attenuated by inhibitors of the protein kinase CK2 independent of heme oxygenase activity

The heme oxygenase (HO) enzymes catalyze the rate-limiting step of heme breakdown, and may accelerate oxidative injury to neurons exposed to heme or hemoglobin. HO-1 and HO-2 are activated in vitro by the phos-phatidylinositol 3-kinase (PI3K)/Akt and protein kinase C (PKC)/CK2 pathways, respectively. The present study tested the hypotheses that CK2, PKC, and PI3K inhibitors would reduce both HO activity and neuronal vulnerability to hemoglobin in murine cortical cultures. Oxidative cell injury was quantified by LDH release and malondialdehyde assays. HO activity was assessed by carbon monoxide assay. Consistent with prior observations, treating primary cortical cultures with hemoglobin for 16h resulted in release of approximately half of neuronal LDH and a seven-fold increase in malondialdehyde. Both endpoints were significantly reduced by the CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), and by the PKC inhibitor GF109203X; the PI3K inhibitors LY294002 and wortmannin had no effect. None of these inhibitors altered basal HO activity. The 1.9-fold activity increase observed after hemoglobin treatment was largely prevented by LY294002 and LY303511, a structural analog of LY294002 that does not inhibit PI3K activity. It was not reduced by wortmannin, TBB or GF109203X. These results suggest that the protective effect of CK2 and PKC inhibitors in this model is not dependent on reduction in HO activity. In this culture system that expresses both HO-1 and HO-2, HO activity does not appear to be primarily regulated by the PKC/CK2 or PI3K pathways.     

Elevation of heme oxygenase‐1 by proteasome inhibition affords dopaminergic neuroprotection

Postmortem studies have shown that heme oxygenase‐1 (HO‐1) immunoreactivity is increased in patients with Parkinson disease. HO‐1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO‐1 content. MG‐132, a proteasome inhibitor, increased the amount of HO‐1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT‐PCR analysis revealed that lactacystin upregulated HO‐1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter‐driven expression of Flag‐HO‐1 protein and resulted in an accumulation of ubiquitinated Flag‐HO‐1 in Flag‐HO‐1‐overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag‐HO‐1 protein was slowed by MG‐132. Next, the function of HO‐1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6‐hydroxydopamine (6‐OHDA)‐induced toxicity and this neuroprotection was abrogated by co‐treatment with zinc protoporphyrin IX, a HO‐1 inhibitor. Furthermore, 6‐OHDA‐induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO‐1‐catalyzed degradation of heme. These results suggest that mesencephalic HO‐1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection. © 2010 Wiley‐Liss, Inc.     

Heme oxygenase attenuated angiotensin II-mediated increase in cyclooxygenase activity and decreased isoprostane F2alpha in endothelial cells

Heme oxygenase (HO) regulates cellular heme levels and catalyzes the formation of bilirubin and carbon monoxide (CO). We hypothesized that the status of the endothelial HO system influences the angiotensin (Ang) II-induced increase in the endothelial cell (EC) production of PGE2, eicosanoids which modulate the vascular actions of Ang II. In this study, we investigated the effect of interventions that suppress HO activity or induce HO-1 gene expression on Ang II-mediated increase in PGE2 in cultures of human microvessel endothelial cells (EC). Incubation of EC with Ang II (100 ng/ml) for 24 h increased the levels of PGE2 in the culture media. This effect of Ang II on prostaglandin production by EC was attenuated in cells treated with heme, but was magnified in cells treated with the HO inhibitor, Stannis mesoporphyrin (SnMP). Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene attenuated heme and Ang II-induced prostaglandin synthesis. We also investigated the physiological significance of human HO-1 overexpression on attenuation of Ang II-mediated oxidative stress. Decreases in COMET levels were found in EC transduced with the HO-1 gene. These results indicate that overexpression of the HO system in EC exerts an inhibitory influence on Ang II-induced synthesis of prostaglandins and attenuates DNA damage caused by exposure to Ang II.     

Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo

Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE), which is a major complication in acute and chronic liver failure and other hyperammonemic states. The molecular mechanisms underlying ammonia neurotoxicity and the functional consequences of ammonia on gene expression in astrocytes are incompletely understood. Using cDNA array hybridization technique we identified ammonia as a trigger of heme oxygenase-1 (HO-1) mRNA levels in cultured rat astrocytes. As shown by Northern and Western blot analysis, HO-1 mRNA levels were upregulated by ammonia (0.1-5 mmol/L) after 24 h and protein expression after 72 h in astrocytes. These ammonia effects on HO-1 are probably triggered to a minor extent by ammonia-induced glutamine synthesis or by astrocyte swelling, because HO-1 expression was not inhibited by the glutamine synthetase inhibitor methionine sulfoximine (which abrogated ammonia-induced cell swelling in cultured astrocytes), and ammonia-induced HO-1 expression could only partly be mimicked by hypoosmotic astrocyte swelling. Hypoosmotic (205 mOsm/L) exposure of astrocytes led even to a decrease in HO-1 mRNA levels within 4 h, whereas hyperosmotic (405 mOsm/L) exposure increased HO-1 mRNA expression. After 24 h, hypoosmolarity slightly raised HO-1 mRNA expression. Taurine and melatonin diminished ammonia-induced HO-1 mRNA or protein expression, whereas other antioxidants (dimethylthiourea, butylated hydroxytoluene, N-acetylcysteine, and reduced glutathione) increased HO-1 mRNA levels under ammonia-free conditions. An in vivo relevance is suggested by the finding that increased HO-1 expression occurs in the brain cortex from acutely ammonia-intoxicated rats. It is concluded that ammonia-induced HO-1 expression may contribute to cerebral hyperemia in hyperammonic states.     

Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice

Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1(+) and GLT1(+) cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1(+) and GLT1(+) astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.     

Heme oxygenase attenuated angiotensin II-mediated increase in cyclooxygenase activity and decreased isoprostane F2α in endothelial cells

Heme oxygenase (HO) regulates cellular heme levels and catalyzes the formation of bilirubin and carbon monoxide (CO). We hypothesized that the status of the endothelial HO system influences the angiotensin (Ang) II-induced increase in the endothelial cell (EC) production of PGE₂, eicosanoids which modulate the vascular actions of Ang II. In this study, we investigated the effect of interventions that suppress HO activity or induce HO-1 gene expression on Ang II-mediated increase in PGE₂ in cultures of human microvessel endothelial cells (EC). Incubation of EC with Ang II (100 ng/ml) for 24 h increased the levels of PGE₂ in the culture media. This effect of Ang II on prostaglandin production by EC was attenuated in cells treated with heme, but was magnified in cells treated with the HO inhibitor, Stannis mesoporphyrin (SnMP). Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene attenuated heme and Ang II-induced prostaglandin synthesis. We also investigated the physiological significance of human HO-1 overexpression on attenuation of Ang II-mediated oxidative stress. Decreases in COMET levels were found in EC transduced with the HO-1 gene. These results indicate that overexpression of the HO system in EC exerts an inhibitory influence on Ang II-induced synthesis of prostaglandins and attenuates DNA damage caused by exposure to Ang II.     

Heme oxygenase-1 (HSP-32) and heme oxygenase-2 induction in neurons and glial cells of cerebral regions and its relation to iron accumulation after focal cortical photothrombosis

Cerebral ischemic injury results in the liberation of heme from degenerating heme-containing proteins. The neurotoxic heme is usually detoxified by the constitutive heme oxygenase-2 (HO-2) and its inducible isoform HO-1(heat shock protein 32) resulting in the formation of biliverdin which becomes reduced to bilirubin, carbon monoxide (CO), and iron. Biliverdin and bilirubin have antioxidative properties whereas CO is discussed as a signaling molecule. Iron if it remains free could catalyze Haber--Weiss and Fenton reactions causing the formation of highly toxic radicals. We have studied the alterations of cerebral HO-2 and HO-1 in relation to iron accumulations after defined cortical photothrombosis within the hindlimb area of the rat. HO-2 immunohistochemistry showed that the number of HO-2-positive neurons in most perilesional regions remained constant. However, much stronger systemic immunoreactivity for HO-2 was observed between days 1 and 7 postlesion. For HO-1 a systemic increase of immunoreactivity occurred also between days 1 and 7. In addition HO-1-positive astrocytes and microglia appeared as early as 4 h postlesion and increased up to day 3 followed by a sharp decline toward day 14 within the injured hemisphere. HO-1-positive astrocytes and microglia occurred in ipsilateral cortex, corpus callosum, hippocampus, striatum, and thalamic nuclei. Additionally an increase of HO-1 in myelin-associated globulin-positive oligodendrocytes was found in ipsilateral and contralateral cortex. Next to the lesion iron accumulation occurred after day 3 and increased strongly toward day 14 at times when HO-1 and -2 had decreased, suggesting that HO activity does not directly contribute to postlesional iron deposition.     

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.     

Reduced HO-1 protein expression is associated with more severe neurodegeneration after transient ischemia induced by cortical compression in diabetic Goto-Kakizaki rats.

Pronounced hyperglycemia provoked by extradural compression (EC) of the sensorimotor cortex was recently described in the non-insulin dependent Goto-Kakizaki (GK) diabetic rat. Compared with control Wistar rats, GK rats exhibited more extensive brain damage after cortical ischemia at 48 h of reperfusion (Moreira et al, 2007). We hypothesized that the enhanced brain injury in GK rats could be caused by differential regulation of the heme degrading enzyme heme oxygenase (HO)-1, known to interact with the expression of other target genes implicated in antioxidant defense, inflammation and neurodegeneration, such as superoxide dismutase (SOD)-1, -2, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-alpha (TNFalpha). At 48 h after ischemia, relative mRNA expression of such target genes was compared between ipsilateral (compressed) and contralateral (uncompressed) hemispheres of GK rats, along with baseline comparison of sham, uncompressed GK and Wistar rats. Immunohistochemistry was performed to detect cellular and regional localization of HO-1 at this time point. Baseline expression of HO-1, iNOS, and TNFalpha mRNA was increased in the cortex of sham GK rats. GK rats showed pronounced hyperglycemia during EC and transient attenuation of regional cerebral blood flow recovery. At 48 h after reperfusion, HO-1 mRNA expression was 7- to 8-fold higher in the ischemic cortex of both strains, being the most upregulated gene under study. Heme oxygenase-1 protein expression was significantly reduced in diabetic rats and was found in perilesional astrocytes and rare microglial cells, in both strains.The reduced HO-1 protein expression in GK rats at 48 h after reperfusion combined with more extensive neurodegeneration induced by EC, provides further in vivo evidence for a neuroprotective role of HO after brain ischemia.     

Protective effects of heme-oxygenase expression in cyclosporine A--induced injury

Cyclosporine A (CsA) is the immunosuppressant of first choice in allotransplantation. Its use is associated with side effects of nephrotoxicity and neurotoxicity, which are among the most prominent. This study was undertaken to explore whether expression and activity of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, is altered in a rat model of CsA-induced injury. Male Sprague Dawley rats were divided into four groups and treated for 21 days. Group I (control) was injected with olive oil (vehicle), group II with CsA (15 mg/kg/day), group III with CsA and the HO inhibitor stannous mesoporphyrin (SnMP) (30 micromol/kg/day) and group IV with one dose of the HO inducer cobalt protoporphyrin (CoPP) 5 mg/100 or heme (10 mg/kg body weight), three days after onset of CsA treatment. Renal tissue was processed for light microscopy, and for HO-1 enzyme activity, assay and for Western blot analysis. In CsA-treated rats there was histological evidence of tubulointerstitial scarring. HO-1 was undetectable in CsA-treated rats compared to control while there was no change in HO-2. In animals treated with a combination of CsA and SnMP, HO-1 activity was further reduced. In animals treated with a combination of CsA and CoPP, HO-1 protein levels were partially restored. These observations indicate that downregulation of HO-1 expression by CsA could be one mechanism underlying CsA-induced toxicity. The CsA-induced decrease in HO-1 expression is partial and restorable, and attempts to preserve HO levels may attenuate CsA toxicity.     

Differential upregulation of heme oxygenase-1 (HSP32) in glial cells after oxidative stress and in demyelinating disorders

Oxidative stress is implicated in the pathogenesis of demyelinating disorders and inflammatory responses. Heme oxygenase-1 (HO-1; HSP32) is a small heat shock protein (HSP) with enzymatic activity, which is inducible by oxidative stress. In this study we analyzed autopsy and biopsy brain samples of patients with multiple sclerosis (MS) and ADEM (acute disseminated leucoencephalomyelits) and spinal cord lesions of mouse EAE (experimental autoimmune encephalomyelitis), which was actively induced by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide, for the presence of HO-1. HO-1 was observed in glial cells during different stages: (1) during acute phases of mainly inflammatory diseases (EAE and ADEM) expression of HO-1 was prominent in microglia/macrophages and astrocytes, and upregulation correlated with inflammation, and (2) in early MS lesions HO-1 was expressed in oligodendrocytes. Furthermore, in glial cell cultures, we can show that upregulation of HO-1 in oligodendrocytes was paralleled by severe morphological damage. Oligodendrocytes underwent apoptotic cell death at a concentration of hydrogen peroxide (50–200 μM) which did not affect astrocytes or microglia. Using oligodendroglial OLN-93 cells, we demonstrate that oxidative stress led to mitochondrial impairment and the disorganization of the microtubule network. Zinc protoporphyrin, an inhibitor of HO-1, augmented the cytotoxic consequences of hydrogen peroxide in OLN-93 cells. Hence, the presence of HO-1 in EAE, ADEM, and MS points to the involvement of oxidative stress and a role of HO-1 in the pathogenesis of the diseases. The data suggest that stress-induced HO-1 initially plays a protective role, while its chronic upregulation, might contribute to oligodendroglial cell death rather than providing protection.