Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes

Increased heme oxygenase (HO)-1 activity attenuates endothelial cell apoptosis and decreases superoxide anion (O2-) formation in experimental diabetes by unknown mechanisms. We examined the effect of HO-1 protein and HO activity on extracellular SOD (EC-SOD), catalase, O2-, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) levels and vascular responses to ACh in control and diabetic rats. Vascular EC-SOD and plasma catalase activities were significantly reduced in diabetic compared with nondiabetic rats (P < 0.05). Upregulation of HO-1 expression by intermittent administration of cobalt protoporphyrin, an inducer of HO-1 protein and activity, resulted in a robust increase in EC-SOD but no significant change in Cu-Zn-SOD. Administration of tin mesoporphyrin, an inhibitor of HO-1 activity, decreased EC-SOD protein. Increased HO-1 activity in diabetic rats was associated with a decrease in iNOS but increases in eNOS and plasma catalase activity. On the other hand, aortic ring segments from diabetic rats exhibited a significant reduction in vascular relaxation to ACh, which was reversed with cobalt protoporphyrin treatment. These data demonstrate that an increase in HO-1 protein and activity, i.e., CO and bilirubin production, in diabetic rats brings about a robust increase in EC-SOD, catalase, and eNOS with a concomitant increase in endothelial relaxation and a decrease in O2-. These observations in experimental diabetes suggest that the vascular cytoprotective mechanism of HO-1 against oxidative stress requires an increase in EC-SOD and catalase.     

上调血红素氧合酶1对糖尿病心肌梗死大鼠心功能的远期影响

目的：研究上调血红素氧合酶1（HO-1）的表达对糖尿病心肌梗死大鼠心功能的影响及其机制。方法：将60只成年Wistar雄性大鼠随机分为5组（n=12）,分别为假手术组、糖尿病组、模型组、诱导剂组、抑制剂组。心梗造模后次日开始给药,1次／周,持续6周,术后28周,应用心脏超声、经颈动脉左心室内插管术等方法观察HO-1诱导剂钴原卟啉（CoPP）及HO活性抑制剂锡中卟啉（SnMP）干预后对心室重构及心功能各项指标的远期影响;测定血糖（GLU）、总胆红素（TB）、C反应蛋白（CRP）、血清肌酐（cr）、转氨酶（ALT）等指标;采用ELISA测定白介素-6（IL-6）、肿瘤坏死因子（TNE）、一氧化氮（NO）、前列环素（PG12）、脂联素、超敏CRP（HsCRP）等水平。结果：应用CoPP上调HO-1水平,能够改善糖尿病心梗大鼠左心室压力最大变化速率、左室射血分数、左室短轴缩短率,缩小左室舒张末期内径,升高血清胆红素、一氧化氮和前列环素水平,提高心肌组织磷酸化的内皮型一氧化氮合酶（peNOS）、磷酸化的活化蛋白激酶（pAkt）、磷酸化的腺苷活化的蛋白激酶（pAMPK）的表达,降低血清TNF-α、hs-CRP水平。使用SnMP后,能够阻断CoPP的上述作用。结论：上调HO-1通过peNOS、pAMPK途径能够长期地改善血管内皮功能,抑制炎症反应,提升血清胆红素等,有效抑制心室重塑,改善梗死后糖尿病大鼠远期的心功能。     

Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide. Carbon monoxide inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. We reported HO-1-mediated endothelial dysfunction in Dahl salt-sensitive hypertension. Previous studies suggested that salt-sensitive hypertensive rats, but not spontaneously hypertensive rats (SHR), display endothelial dysfunction. This study examines the hypothesis that HO-1-mediated arteriolar endothelial dysfunction develops in deoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA) rats, but not in SHR. Uninephrectomized (isoflurane anesthesia) male Sprague-Dawley rats received DOCA injections and saline drinking solution for 4 wk. Rats subjected to sham surgery received vehicle injections and tap water. Blood pressure was elevated in DOCA rats and SHR compared with sham and Wistar-Kyoto (WKY) groups. Aortic HO-1 expression and blood carboxyhemoglobin levels were elevated in the DOCA group, but not in SHR. In isolated gracilis muscle arterioles, ACh caused concentration-related vasodilation in all groups, with attenuated maximum responses in DOCA, but not in SHR, arterioles. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, restored ACh-induced responses in DOCA arterioles to sham levels. ACh responses remained the same in SHR and WKY arterioles after chromium mesoporphyrin treatment. These data show that HO-1 levels and activity are increased and arteriolar responses to ACh are decreased in DOCA rats, but not in SHR. Furthermore, in DOCA arterioles, an inhibitor of HO restores ACh-induced vasodilation to sham levels. These results suggest that elevated HO-1 levels and activity, not resulting from hypertension per se, contribute to endothelial dysfunction in DOCA rats.     

The Induction of Heme Oxygenase 1 Decreases Painful Diabetic Neuropathy and Enhances the Antinociceptive Effects of Morphine in Diabetic Mice

Painful diabetic neuropathy is a common complication of diabetes mellitus which is poorly controlled by conventional analgesics. This study investigates if treatment with an heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX (CoPP), could modulate the allodynia and hyperalgesia induced by diabetes and enhanced the antinociceptive effects of morphine. In a diabetic mice model induced by the injection of streptozotocin (STZ), we evaluated the antiallodynic and antihyperalgesic effects produced by the intraperitoneal administration of 5 and 10 mg/kg of CoPP at several days after its administration. The antinociceptive actions produced by the systemic administration of morphine alone or combined with CoPP were also evaluated. In addition, the effects of CoPP treatment on the expression of HO-1, the microglial activation marker (CD11b/c), the inducible nitric oxide synthase (NOS2) and μ-opioid receptors (MOR), were also assessed. Our results showed that the administration of 10 mg/kg of CoPP during 5 consecutive days completely blocked the mechanical and thermal hypersensitivity induced by diabetes. These effects are accompanied by the increased spinal cord, dorsal root ganglia and sciatic nerve protein levels of HO-1. In addition, the STZ-induced activation of microglia and overexpression of NOS2 in the spinal cord were inhibited by CoPP treatment. Furthermore, the antinociceptive effects of morphine were enhanced by CoPP treatment and reversed by the administration of an HO-1 inhibitor, tin protoporphyrin IX (SnPP). The spinal cord expression of MOR was also increased by CoPP treatment in diabetic mice. In conclusion, our data provide the first evidence that the induction of HO-1 attenuated STZ-induced painful diabetic neuropathy and enhanced the antinociceptive effects of morphine via inhibition of microglia activation and NOS2 overexpression as well as by increasing the spinal cord levels of MOR. This study proposes the administration of CoPP alone or combined with morphine as an interesting therapeutic approach for the treatment of painful diabetic neuropathy.     

Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.     

Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary syndrome

During hepatopulmonary syndrome caused by liver cirrhosis, pulmonary endothelial nitric oxide (NO) synthase (NOS) expression and NO production are increased. Increased NO contributes to the blunted hypoxic pressor response (HPR) during cirrhosis and may induce heme oxygenase-1 (HO-1) expression and carbon monoxide (CO) production, exacerbating the blunted HPR. We hypothesized that NO regulates the expression of HO-1 during cirrhosis, contributing to hepatopulmonary syndrome. Cirrhosis was induced in rats by common bile duct ligation (CBDL). Rats were studied 2 and 5 wk after CBDL or sham surgery. Lung HO-1 expression was elevated 5 wk after CBDL. Liver HO-1 was increased at 2 wk and remained elevated at 5 wk. In catheterized rats, the blunted HPR was partially restored by HO inhibition. Rats treated with the NOS inhibitor N(G)-nitro-L-arginine methyl ester for the entire 2- or 5-wk duration had normalized HO-1 expression and HPR. These data provide in vivo evidence for the NO-mediated upregulation of HO-1 expression and support the concept that hepatopulmonary syndrome is multifactorial, involving not only NO, but also HO-1 and CO.     

Heme oxygenase-derived carbon monoxide promotes arteriolar endothelial dysfunction and contributes to salt-induced hypertension in Dahl salt-sensitive rats

Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. After 4 wk of high-salt diet, Dahl salt-sensitive (Dahl-S) rats display hypertension, increased vascular HO-1 expression, and attenuated vasodilator responses to ACh that can be completely restored by acute treatment with an inhibitor of HO. In this study, we examined the temporal development of HO-mediated endothelial dysfunction in isolated pressurized first-order gracilis muscle arterioles, identified the HO product responsible, and studied the blood pressure effects of HO inhibition in Dahl-S rats on a high-salt diet. Male Dahl-S rats (5-6 wk) were placed on high-salt (8% NaCl) or low-salt (0.3% NaCl) diets for 0-4 wk. Blood pressure increased gradually, and responses to an endothelium-dependent vasodilator, ACh, decreased gradually with the length of high-salt diet. Flow-induced dilation was abolished in hypertensive Dahl-S rats. Acute in vitro pretreatment with an inhibitor of HO, chromium mesoporphyrin (CrMP), restored endothelium-dependent vasodilation and abolished the differences between groups. The HO product CO prevented the restoration of endothelium-dependent dilation by CrMP. Furthermore, administration of an HO inhibitor lowered blood pressure in Dahl-S rats with salt-induced hypertension but did not do so in low-salt control rats. These results suggest that hypertension and HO-mediated endothelial dysfunction develop gradually and simultaneously in Dahl-S rats on high-salt diets. They also suggest that HO-derived CO underlies the impaired endothelial dysfunction and contributes to hypertension in Dahl-S rats on high-salt diets.     

Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress

Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS(-/-) mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.     

Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background

Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated.

Methodology/Principal Findings

We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression.

Conclusions/Significance

This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain.     

Differential effect of cobalt protoporphyrin on distributions of heme oxygenase in renal structure and on blood pressure in SHR.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, releasing iron and carbon monoxide (CO). Thus, HO not only controls the availability of heme for the synthesis of hemeproteins but also generates CO, which binds to the heme moiety of hemoproteins, thereby affecting their enzymatic activity. The present study was undertaken to explore changes in the relative expression of renal HO-1 and HO-2 in response to modulators and the effect on blood pressure regulation in spontaneously hypertensive rats (SHR). Immunohistochemistry confirmed a cobalt protoporphyrin (CoPP)-mediated increase in HO-1 protein. After a single injection of CoPP (5 mg/100 gram body weight) in 7-week-old SHR, blood pressure significantly decreased (p<0.01) while renal HO activity increased 6-fold over controls. CoPP pretreatment deceased the levels of the renal cytochrome P450-derived arachidonic acid metabolite, 20-HETE, a powerful vasoconstrictor, by 65% in renal tissue. Western blot analysis demonstrated that CoPP significantly increased HO-1 protein expression in the cortex and outer medulla and, to a lesser degree, in the inner medulla of the rat kidney. HO-2 was constitutively expressed in all parts of the kidney, and did not significantly change after treatment with CoPP. These results indicate that selective induction of cortical and outer medullary HO-1 is associated with a decrease in 20-HETE and blood pressure, suggesting an important role for HO-1 activity in the regulation of urine volume, electrolyte excretion and blood pressure.     

Long-Lasting Expression of HO-1 Delays Progression of Type I Diabetes in NOD Mice

Heme oxygenase-1 (HO-1) is crucial in regulating oxidative injury. The present study was designed to assess whether HO-1 upregulation by cobalt protoporphyrin IX (CoPP) moderates or prevents the diabetic state in non-obese diabetic (NOD) mice, an animal model for Type 1 diabetes (T1D). HO-1 expression and HO activity were upregulated in the pancreas by the intermittent administration of CoPP. This was associated with decreases in blood glucose and pancreatic O2-, but increased pAKT and BcL-XL and cell survival. A considerable number of beta cells were preserved in the islets of CoPP-treated NOD mice, while none were found in untreated diabetic mice. The number of CD11c+ dendritic cells was decreased in the pancreas of CoPP-treated NOD mice (p     

Improved myocardial perfusion in chronic diabetic mice by the up‐regulation of pLKB1 and AMPK signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up‐regulating pAMPK‐pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild‐type and streptozotocin (STZ)‐treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO‐1, 3‐nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time‐dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L‐NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK‐3 expression. Cobalt protoporphyrin treatment to induce HO‐1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up‐regulation of HO‐1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK‐3, and peNOS levels and a decrease in myocardial superoxide and 3‐nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes. J. Cell. Biochem. 109: 1033–1044, 2010. © 2010 Wiley‐Liss, Inc.     

High-fat diet exacerbates renal dysfunction in SHR: reversal by induction of HO-1-adiponectin axis

High-dietary fat intake is a major risk factor for development of metabolic and cardiovascular-renal dysfunction including obesity, coronary artery disease, hypertension, and chronic renal failure. We examined the effect of a high-fat diet on renal function and morphology in spontaneously hypertensive rats (SHR), a phenotype designed to mimic metabolic syndrome. High-fat diet induced increase (P < 0.05) in blood pressure, body weight, and renal lipid deposition in these rats. This increase in body weight was accompanied by elevations (P < 0.05) of blood glucose and low-density lipoprotein (LDL) levels, a decrease (P < 0.05) in adiponectin and increases (P < 0.05) in plasma monocyte chemotactic protein-1 (MCP-1) along with renal macrophage infiltration. These pathophysiological perturbations were attenuated (P < 0.05) by heme oxygenase-1 (HO-1) induction by treatment with cobalt protoporphyrin (CoPP). Further effects of CoPP included increased (P < 0.05) renal expression of adiponectin along with enhancement (P < 0.05) of pAKT, pAMPK, and p-eNOS in SHRs fed a high-fat diet. Prevention of such beneficial effects of CoPP by the concurrent administration of the heme-HO inhibitor stannous mesoporphyrin (SnMP) corroborates the role of HO system in mediating such effects. Taken together, our results demonstrate that high-fat diet induces a metabolic syndrome-like phenotype in hypertensive rats, which is amenable to rescue by increases in HO-1- and adiponectin-dependent pathways.     

Effects of hyperthermia pretreatment on expression of heme oxygenase-1 and nitric oxide synthase in rats subjected to experimental anaphylactic shock

Previous studies have shown that hyperthermia pretreatment results in an attenuation of increased vascular leakage in rats subjected to experimental anaphylactic shock. It is known that both nitric oxide synthase (NOS) and heme oxygenase-1 (HO-1) play a role in the maintenance of microvascular integrity. In the study, we investigated the effect of hyperthermia pretreatment on mRNA expression of endothelial NOS (eNOS), inducible NOS (iNOS), and HO-1 in heated or nonheated rats subjected to anaphylactic shock using a semi-quantitative RT-PCR. Protein contents of eNOS and HO-1 in tissue were also assayed. Plasma nitrite and nitrate before and after induction of anaphylactic shock were quantified using a NO analyzer. The heated, anaphylactic rats showed a significant increase of HO-1 mRNA expression in heart as compared to both non-heated, anaphylactic and control rats. HO-1 protein contents in both heart and lung tissues in the heated, anaphylactic rats were significantly higher than both non-heated, anaphylactic and control rats. Protein contents of eNOS in various tissues appeared to be the same among groups. No significant change of iNOS mRNA expression was detected among groups. Plasma nitrite and nitrate before and after anaphylactic treatment appeared to be the same among groups. These data suggest that reduction of anaphylactic hypotension by hyperthermia pretreatment in rats subjected to anaphylactic shock may be resulted from over-expression of HO-1 rather than NOS in various tissues.     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Heme oxygenase expression in selected regions of term human placenta

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.     

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer’s disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H₂O₂-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.     

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors.

Human umbilical vein endothelial cells (HUVECs) were exposed to low (5?mmol/l) and high (25?mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10?μmol/l), HO antagonist (SnPPIX, 10?μmol/l), and NO synthase blocker (l-NAME, 200?μmol/l) with or without NO donor (arginine, 1?mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF).

In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Neuroprotective role of heme-oxygenase 1 against iodoacetate-induced toxicity in rat cerebellar granule neurons: Role of bilirubin

Heme oxygenase (HO) catalyses the breakdown of heme to iron, carbon monoxide and biliverdin, the latter being further reduced to bilirubin. A protective role of the inducible isoform, HO-1, has been described in pathological conditions associated with the production of reactive oxygen species (ROS). The aim of this study was to investigate the role of HO-1 in the neurotoxicity induced by iodoacetate (IAA) in primary cultures of cerebellar granule neurons (CGNs). IAA, an inhibitor of the glycolysis pathway, reduces cell survival, increases ROS production and enhances HO-1 expression in CGNs. Furthermore, the induction of HO-1 expression by cobalt protoporphyrin (CoPP) prevented cell death and ROS production induced by IAA, whereas the inhibition of HO activity with tin mesoporphyrin exacerbated the IAA-induced neurotoxicity. The protective effect elicited by CoPP was reproduced by bilirubin addition, suggesting that this molecule may be involved in the protective effect of HO-1 induction in this experimental model.