Current Neurovascular Research, Vol. 2, No. 2, 2005
Contents
Introduction from the Guest Editor: The Many Facets of
Heme Oxygenase Pp.101-101
N.G. Abraham
Original Articles
Heme Oxygenase Overexpression Attenuates Glucose-Mediated
Oxidative Stress in Quiescent Cell Phase: Linking Heme to Hyperglycemia
Complications Pp.103-111
David Sacerdoti, Rafal Olszanecki, Giovanni Li Volti,
Claudia Colombrita, Giovanni Scapagnini and Nader G. Abraham
Heme Oxygenase-1 in Vascular Smooth Muscle Cells
Counteracts Cardiovascular Damage Induced by Angiotensin II Pp.113-120
Toshisuke Morita, Tomihiko Imai, Takao Sugiyama,
Shigehiro Katayama and Gen Yoshino
Heme Oxygenase-2 Protects Against Glutathione
Depletion-induced Neuronal Apoptosis Mediated by Bilirubin and Cyclic GMP Pp.121-131
Jijun Chen, Yajun Tu, Erin C. Connolly and Gabriele V.
Ronnett
Increased Pulmonary Heme Oxygenase-1 and d-Aminolevulinate Synthase Expression in
Monocrotaline-Induced Pulmonary Hypertension Pp.133-139
Tatsuo Iwasaki, Toru Takahashi, Hiroko Shimizu, Emiko
Ohmori, Taro Morimoto, Masahito Kajiya, Mamoru Takeuchi, Kiyoshi Morita, Reiko
Akagi and Fumihiko Kajiya
Propofol Inhibits Caspase-3 in Astroglial Cells: Role of
Heme Oxygenase-1 Pp.141-148
Rosaria Acquaviva, Agata Campisi, Giuseppina Raciti,
Roberto Avola, Maria Luisa Barcellona, Luca Vanella and Giovanni Li Volti
Plasma Infusions into Porcine Cerebral White Matter
Induce Early Edema, Oxidative Stress, Pro-Inflammatory Cytokine Gene Expression
and DNA Fragmentation: Implications for White Matter Injury with Increased
Blood-
Brain-Barrier Permeability Pp.149-155
Kenneth R. Wagner, Christopher Dean, Shauna Beiler, David
W. Bryan, Benjamin A. Packard, A. George Smulian, Michael J. Linke and
Gabrielle M. de Courten-Myers
Brief Communications
Protective Effects of Heme-Oxygenase Expression in
Cyclosporine A -Induced Injury Pp.157-161
Rita Rezzani, Luigi Rodella, Rossella Bianchi, Alvin I.
Goodman and Elias A. Lianos
Characterization of Heme Oxygenase in Adult Rodent
Platelets Pp.163-168
Hean Zhuang, Marguerite T. Littleton-Kearney, and Sylvain
Dore
Adrenergic Modulation of Dendritic Cells Function:
Relevance for the Immune Homeostasis Pp.169-173
Georges J.M. Maestroni
Melatonin Prevents Hyperhomocysteinemia and Neural Lipid
Peroxidation Induced by Methionine Intake Pp.175-178
Mounir Bouzouf, Francisco Martinez-Cruz, Patrocinio
Molinero, Juan M Guerrero and Carmen Osuna
Review Article
A Second Look into the Oxidant Mechanisms in Alzheimer’s
Disease Pp.179-184
Paula I. Moreira, Catarina R. Oliveira, Maria S. Santos,
Akihiko Nunomura, Kazuhiro Honda, Xiongwei Zhu, Mark A. Smith, George Perry
Abstracts
[Back to top]
Introduction from the Guest Editor: The Many Facets of Heme Oxygenase
N.G. Abraham
This volume of hot topics is dedicated to Attallah Kappas, M.D., former Vice President of The Rockefeller University Hospital and Professor of Pharmacology, for his contribution in developing an overall approach to understanding the significant role of the heme oxygenase system. Dr. Kappas’ laboratory was the first to purify rat heme oxygenase (HO) and describe the molecular weight of this fascinating protein. HO can be considered as both a bad guy and a good guy. It can be bad protein, such as in hyperbilirubinemia or, more commonly, jaundice. Dr. Kappas’ laboratory was the first to develop HO inhibitors, such as SnMP. Now, SnMP is the preferred therapy of choice for hyperbilirubinemia. In collaboration with his peers, including myself, Dr. Kappas has helped to demonstrate that HO can also be a good protein. It is an important regulator for cell cycle progression and its induction in diabetes renders the endothelium to be cytoprotective. HO has been shown to be important in attenuating the overall production of reactive oxygen species (ROS) through its ability to degrade heme and to produce carbon monoxide (CO) and bilirubin. Excess free heme catalyzes the formation of ROS, which may lead to endothelial cell (EC) dysfunction as seen in numerous pathological conditions, such as neurovascular disease, degenerative disease and hypertension as well as ischemia/reperfusion injury. The upregulation of HO-1 through the use of pharmaceutical agents such as metalloporphyrins and through site- and organ-specific targeted gene transfer has become a powerful tool for studying the role of this enzyme in the treatment of various cardiovascular diseases and diabetes. The ability to upregulate the HO-1 gene using gene transfer or pharmacological agents may offer a therapeutic strategy for treating neurovascular and cardiovascular disease in the future. This volume discusses, for the first time, the implications of HO-1 during the early stages of cardiovascular system development and suggests that HO-1 gene expression may prevent the onset of different pathological conditions. Furthermore, it provides the basis for future studies, at the basic science level, regarding the effect of HO-1-derived CO and bilirubin to bypass the induction of HO-1. We are convinced that this volume will be a useful reference for scientists and investigators who are interested in the study of the HO system.
[Back to top]
Heme Oxygenase Overexpression Attenuates Glucose-Mediated Oxidative Stress in
Quiescent Cell Phase: Linking Heme to Hyperglycemia Complications
David Sacerdoti, Rafal Olszanecki, Giovanni Li Volti, Claudia Colombrita, Giovanni Scapagnini and Nader G. Abraham
Heme oxygenase (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against glucose induced oxidative injury. The purpose of this study was to examine the role of human HO-1 in attenuating glucose-mediated oxidative stress. We investigated the effect of high ambient glucose (15, 33 and 66 mM) on HO-1 gene expression in endothelial cells grown in a serum deprived media compared to the effect of glucose on exponentially grown cells (10% FBS). High glucose at 15 and 33 mM caused significant inhibition of HO-1 protein and activity in G0/G1 and in cells exponentially grown. Glucose concentration at 66mM caused a significant increase in HO-1. Addition of heme (10mM) increased HO-1 protein and bilirubin formation in G0/G1, in a time dependent manner peaking at 16h. Glucose attenuated heme mediated increase in HO-1 proteins. RT-PCR demonstrated that glucose decreased the levels of HO-1 mRNA in both G0/G1 or cells grown in 10% FBS. The rate of HO-1 induction in response to heme was several fold higher in serum-starved cells compared to cells cultured in 10% FBS. Cells exposed to high glucose for up to 24 h had a significant increase in cellular heme and potentiated heme-mediated increase in generation of superoxide anion and 8-epiisoprostane PGF2a. HO-1 gene transduction prevented glucose-mediated elevation of 8-epi-isoprostane PGF2a. These results imply that expression of HO-1 in G0/G1 cells may be a key player in decreasing cellular heme, associated with increased generation of bilirubin, and in attenuating glucose mediated oxidative stress.
[Back to top] Heme
Oxygenase-1 in Vascular Smooth Muscle Cells Counteracts Cardiovascular Damage
Induced by Angiotensin II
Toshisuke Morita, Tomihiko Imai, Takao Sugiyama,
Shigehiro Katayama and Gen Yoshino
Heme oxygenase (HO) is a microsomal enzyme that catalyzes the degradation of heme into biliverdin, which is subsequently reduced to bilirubin, free iron and carbon monoxide (CO), and induction of heme oxygenase-1 (HO-1) is potentially associated with cellular protection, especially against oxidative insults. Using transgenic mice that overexpress HO-1 (HO-1 Tg) specifically in vascular smooth muscle cells, we investigated the organ-protective effects of HO-1 against angiotensin II (Ang II). Following administration of Ang II and a high- salt diet for 14 days, marked intimal hyperplasia as well as inflammatory changes were observed in coronary arteries of Ang II/salt-treated wild type (Wt) mice. In Wt mice, Ang II/salt loading increased urinary excretion of 8- hydroxydeoxyguanosine (8-OHdG) and 8-lso- Prostaglandin F2 alpha. Cardiac levels of MDA and 4-HAE, markers of lipid peroxidation, and GSSG/GSH were also increased in Wt. mice after Ang II/salt loading, but not in HO-1 Tg mice. Consistently, immunostaining for both 8-0HdG, a marker of oxidative DNA damage, and 3-nitrotyrosine, the metabolites of reactive oxygen species, were apparently increased in the Ang II/salt-treated heart of Wt. mice; however, no significant changes in these responses were detected in HO-1 Tg mice after Ang II/salt loading. These data suggest that increased oxidative stress might be involved in the coronary artery changes induced by Ang II/salt loading. The evidence presented in the current study indicates that vascular HO-1 exerts its protective effect against cardiovascular damage, possibly through the inhibition of oxidative stress.
[Back
to top] Heme Oxygenase-2 Protects Against Glutathione
Depletion-induced Neuronal Apoptosis Mediated by Bilirubin and Cyclic GMP
Jijun Chen, Yajun Tu, Erin C. Connolly and Gabriele V.
Ronnett
Heme oxygenase (HO) enzymes catalyze the breakdown of heme to iron, carbon monoxide (CO), and biliverdin, which is rapidly converted to bilirubin. HO-2 has been implicated in protection against oxidative stress, ischemia, and traumatic brain injury. The neuroprotective effects of HO-2 have been attributed to the generation of bilirubin, which is an important radical scavenger. However, the mechanism by which HO-2 provides protection is unclear. We utilized the olfactory system as a model to define the roles of HO-2 in glutathione depletion-induced oxidative injury, since olfactory receptor neurons (ORNs) express high levels of HO isoforms. We demonstrated that Lbuthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione biosynthesis, lowered glutathione levels and induced apoptosis of ORNs. Despite the presence of HO-1 in ORNs, HO-2 null animals displayed increased levels of neuronal death after BSO treatment compared to wild type mice. Levels of bilirubin and cGMP were also reduced in HO-2 null mice. Primary cultures of ORNs confirmed that the neuroprotective role of HO-2 was mediated by bilirubin and cGMP. Taken together, these results suggest that HO-2 plays a major role in neuroprotection from oxidative stress, an effect that is mediated by cGMP and bilirubin.
[Back to top]
Increased Pulmonary Heme Oxygenase-1 and d-Aminolevulinate
Synthase Expression in Monocrotaline-Induced Pulmonary Hypertension
Tatsuo Iwasaki, Toru Takahashi, Hiroko Shimizu, Emiko
Ohmori, Taro Morimoto, Masahito Kajiya, Mamoru Takeuchi, Kiyoshi Morita, Reiko
Akagi and Fumihiko Kajiya
Monocrotaline (MCT), a pyrrolizidine alkaloid plant toxin, is known to cause pulmonary hypertension (PH) in rats. Recent findings suggest that pulmonary inflammation may play a significant role in the pathogenesis in MCTinduced PH. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, is known to be induced by various oxidative stresses, including inflammation and free heme, and its induction is thought essential in the protection against oxidative tissue injuries. In this study, we examined expression of HO-1 as well as non-specific d-aminolevulinate synthase (ALAS1), the rate-limiting enzyme in heme catabolism and biosynthesis, respectively, in a rat model of PH produced by subcutaneous injection of MCT (60 mg/kg). MCT treatment caused infiltration of inflammatory cells, fibrosis of the interstitium, and pulmonary arterial wall thickening with marked elevation of right ventricular (RV) pressure, which are characteristics of MCT-induced PH. Gene expression of tumor necrosis factor-a (TNF-a) as well as DNA binding activity of nuclear factor-kB (NF-kB) increased at 1 week after MCT treatment, reached a maximum at 2 weeks, and then decreased to the pretreatment level at 3 weeks. HO-1 expression was markedly increased at 1 week, and continued to increase by 3 weeks following MCT treatment, both at transcriptional and protein levels in the mononuclear cells in the lung. ALAS1 mRNA levels in the lung also significantly increased at 2 weeks after MCT treatment. These findings suggest that pulmonary HO-1 expression was presumably induced by proinflammatory cytokine(s) in MCTtreated rats, resulting in the derepression of heme-repressible ALAS1 expression, and that HO-1 induction plays a significant role as an inflammatory factor in this condition.
[Back to top] Propofol
Inhibits Caspase-3 in Astroglial Cells: Role of Heme Oxygenase-1
Rosaria Acquaviva, Agata Campisi, Giuseppina Raciti,
Roberto Avola, Maria Luisa Barcellona, Luca Vanella and Giovanni Li Volti
Several lines of evidence have extensively demonstrated that peroxynitrite plays a pivotal role in Central Nervous System (CNS) injuries. The present study was aimed at elucidating the molecular mechanism by which propofol attenuates peroxynitrite-mediated injury in the brain. Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (SIN-1,3 mM) in the presence or absence of propofol (40 mM, 80 mM and 160 mM). The protective effects of propofol were evaluated by MTT cytotoxicity assay, LDH release, and caspase-3 activation by Western blot analysis. Appropriate propofol concentrations (ranging from 40 mM to 160 mM) significantly increased HO-1 expression and attenuated SIN-1-mediated cytotoxicity and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin-mesoporphirin (SnMP), a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of NF-kB abolished propofol-mediated HO-1 induction, suggesting a possible role for this nuclear transcriptional factor in our experimental conditions. These findings indicate that propofol attenuates peroxynitritemediated apoptosis in astroglial cells, a property that may be relevant in both physiological and pathological processes in the CNS.
[Back to top] Plasma
Infusions into Porcine Cerebral White Matter Induce Early Edema, Oxidative
Stress, Pro-Inflammatory Cytokine Gene Expression and DNA Fragmentation:
Implications for White Matter Injury with Increased Blood-Brain-Barrier
Permeability
Kenneth R. Wagner, Christopher Dean, Shauna Beiler, David
W. Bryan, Benjamin A. Packard, A. George Smulian, Michael J. Linke and
Gabrielle M. de Courten-Myers
Plasma infused into porcine cerebral white matter induces both acute interstitial and delayed vasogenic edema. Edematous white matter contains extracellular plasma proteins and rapidly induces oxidative stress as evidenced by increased protein carbonyl formation and heme oxygenase-1 induction. We tested the hypothesis that edematous white matter would also upregulate pro-inflammatory cytokine gene expression and develop DNA damage. We infused autologous plasma into the frontal hemispheric white matter of pentobarbital-anesthetized pigs. We monitored and controlled physiological variables and froze brains in situ at 1, 4 or 24 hrs. We determined edema volumes by computerassisted morphometry. We measured white matter protein carbonyl formation by immunoblotting, cytokine gene expression by standard RT-PCR methods and DNA fragmentation by agarose gel electrophoresis. White matter edema developed acutely (1 hr) after plasma infusion and increased significantly in volume between 4 and 24 hrs. Protein carbonyl formation also occurred rapidly in edematous white matter with significant elevations (3 to 4-fold) already present at 1 hr. This increase remained through 24 hrs. Pro-inflammatory cytokine gene expression was also rapidly increased at 1 hr post-infusion. Evidence for DNA fragmentation began at 2 to 4 hrs, and a pattern indicative of both ongoing necrosis and apoptosis was robust by 24 hrs. Plasma protein accumulation in white matter induces acute edema development and a cascade of pathochemical events including oxidative stress, pro-inflammatory cytokine gene expression and DNA damage. These results suggest that in diseases with increased blood-brain barrier (BBB) permeability or following intracerebral hemorrhage or traumatic brain injury, interstitial plasma can rapidly damage white matter.
[Back to top] Protective Effects of
Heme-Oxygenase Expression in Cyclosporine A -Induced Injury
Rita Rezzani, Luigi Rodella, Rossella Bianchi, Alvin I.
Goodman and Elias A. Lianos
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 mmol/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 CsAinduced toxicity. The CsA-induced decrease in HO-1 expression is partial and restorable, and attempts to preserve HO levels may attenuate CsA toxicity.
[Back to top] Characterization of
Heme Oxygenase in Adult Rodent Platelets
Hean Zhuang, Marguerite T. Littleton-Kearney, and Sylvain
Dore
Thromboembolism — and its involvement with tissue infarction and ischemic necrosis — continues to be of major importance in the area of vascular biology that affects all areas of clinical medicine. Activated platelets and their aggregations are key initiators in the formation of the thrombus. Several mechanisms have been described to modulate thrombus formation in the circulation, such as prostacyclins and endothelium-derived relaxing factors (the most studied being nitric oxide). Similar to nitrous oxide (NO), carbon monoxide (CO) can modulate guanylate cyclase and has been associated with anti-inflammatory and anti-apoptotic activities. Heme oxygenase (HO), in addition to being the ratelimiting enzyme of CO generation, degrades heme, which is a pro-oxidant/pro-inflammatory and generates the antioxidant molecules biliverdin and bilirubin. HO-2 is generally considered to be enriched in the brain. Here, by studying mouse platelets, we showed that it is highly present in wildtype (WT) animals and not detectable in HO-2 knockout mice. A similar finding was observed in female rats. We also investigated whether modification of estrogen levels (naturally occurring, with age, or surgically) and estrogen replacement would affect intraplatelet HO levels. Under these chronic conditions, HO-1 was barely detectable, while HO-2 was consistently stably expressed at high levels. Further investigation into the functional properties of HO itself, heme degradation, and heme bioactive metabolites remains to be conducted to determine the role of HO on platelet dynamics and on microvasculature.
[Back to top] Adrenergic
Modulation of Dendritic Cells Function: Relevance for the Immune Homeostasis
Georges J.M. Maestroni
Dendritic cells (DC) play a key role in determining the appropriate immune response to invading pathogens and tolerance to self antigens. Here I review the evidence that dendritic cell functions may be tuned by the sympathetic nervous system via the local release of norepinephrine. In the presence of antigens or microbial products, such as agonists for Toll-like receptors 2 and 4, norepinephrine inhibits dendritic cell migration, antigen presentation and T-helper cells type 1 priming. This effect, which is mainly mediated by b-adrenergic receptors in DC and interlukin-10 production, limits potentially damaging reactions and is functional in shaping the appropriate humoral immune response to extracellular pathogens that need antibodies to be neutralized. In addition, the response to contact sensitizers seems to involve a modulation of the local sympathetic activity. Thus, the sympathetic nervous system may play a crucial role in modulating DC function during the innate phase of the immune response. This evidence has many pathophysiological implications and offers new tools for modulating the immune response.
[Back to top] Melatonin Prevents
Hyperhomocysteinemia and Neural Lipid Peroxidation Induced by Methionine Intake
Mounir Bouzouf, Francisco Martinez-Cruz, Patrocinio
Molinero, Juan M Guerrero and Carmen Osuna
This study was designed to determine the protective effect of melatonin treatment against oxidative damage in rat brain induced by hyperhomocysteinemia (Hhcy). Oral administration of methionine and its degradation product, homocysteine (hcy), causes mild to moderate Hhcy. The major end-point of oxidative damage measured in this report was lipid peroxidation (LPO). The levels of malondialdehyde (MDA) were assayed as index of lipid peroxidation. The increase in lipid peroxidation was inhibited by melatonin. Rats were divided into seven groups: one was used as control and each remaining group was supplemented with methionine dissolved and added to the drinking water daily for 4 weeks (0.5, 1, 1.5, 2, 3g /kg BW). Additional groups of rats were given both melatonin (30 mg/kg BW) and methionine in drinking water daily. At the conclusion of the study, MDA levels were significantly increased in the brains of methioninetreated rats compared with control rats, whereas melatonin prevented the increases in MDA levels. Plasma hcy levels in animals treated with melatonin were significantly lower than those of controls. Melatonin lowered plasma hcy levels and could potentially be beneficial in prevention of neurodegeneration caused by mild hyperhomocysteinemia.
[Back to top] A Second Look into
the Oxidant Mechanisms in Alzheimer’s Disease
Paula I. Moreira, Catarina R. Oliveira, Maria S. Santos,
Akihiko Nunomura, Kazuhiro Honda, Xiongwei Zhu, Mark A. Smith, George Perry
Oxidative damage is a major feature of Alzheimer’s disease pathophysiology. Instead of succumbing to these oxidative abnormalities, neurons upregulate antioxidant defenses, which suggest a novel balance in oxidant homeostasis in Alzheimer’s disease. Evidence indicates that in the initial phase of Alzheimer’s disease development, amyloid-b deposition and hyperphosphorylated t are consequences of oxidative stress and function as a primary line of antioxidant defense. However, during the progression of the disease, the antioxidant activity of both agents evolves into pro-oxidant, representing a typical gain-of-function transformation. This transformation is due to an increase in reactive species and a decrease in clearance mechanisms. However, the notion that amyloid-b and hyperphosphorylated t function as protective components in the early stages of Alzheimer’s disease brings into serious question the rationale of current therapeutic strategies aimed to remove both amyloid-b and hyperphosphorylated t.