Human Extracellular Superoxide Dismutase Research Articles

Long-term Suppression of Neurodegeneration in Chronic Experimental Optic Neuritis: Antioxidant Gene Therapy

To test in mice with experimental autoimmune encephalomyelitis (EAE) a strategy designed to treat patients at risk for axonal degeneration and persistent visual loss from optic neuritis and multiple sclerosis. The authors cloned the human extracellular superoxide dismutase (ECSOD) or catalase (CAT) gene into recombinant adenoassociated virus (AAV). Transgene expression was evaluated by immunochemistry of infected RGC-5 cells and after intravitreal injection of AAV-ECSOD or AAV-CAT, or both, into the right eyes of DBA/1J mice. Control cells and left eyes were inoculated with AAV-GFP. Animals were sensitized for EAE, followed by serial contrast-enhanced MRI for 6 months, and then were euthanatized. The effects of ECSOD and CAT modulation on the EAE optic nerve were gauged by computerized analysis of optic nerve volume, myelin fiber area, axonal cell loss, and retinal ganglion cell (RGC) loss. Western blot analysis of infected RGC-5 cells revealed that expression of ECSOD increased 15-fold and that of CAT increased 3.5-fold. One month after intraocular injections, transgene expression increased 4-fold for AAV-ECSOD and 3.3-fold for AAV-CAT. Six months after intraocular injections and EAE sensitization, combination therapy with ECSOD and catalase decreased RGC loss by 29%, optic nerve demyelination by 36%, axonal loss by 44%, and cellular infiltration by 34% compared with the contralateral control eyes inoculated with AAV-GFP. Compared with the normal optic nerve, it limited RGC loss to 9%. Viral-mediated delivery of antioxidant genes provides long-lasting suppression against neuronal and axonal loss associated with permanent visual disability in patients with optic neuritis and multiple sclerosis.

Gene Transfer of Extracellular Superoxide Dismutase Ameliorates Pulmonary Hypertension in Rats

Pulmonary hypertension (PH) is a life-threatening disease, characterized by vascular remodeling and vasoconstriction. Evidence suggests that oxidative stress may contribute to the pathogenesis and/or development of PH. In the present study, we examined whether intratracheal gene transfer of human extracellular superoxide dismutase (EC-SOD) could ameliorate monocrotaline (MCT)-induced PH in rats. MCT-injected rats were intratracheally administered vehicle (MCT group) or an adenovirus encoding beta-galactosidase (Adbetagal group) or human EC-SOD (AdEC-SOD group). After intratracheal gene transfer, EC-SOD was successfully expressed in lung tissue, bronchoalveolar lavage fluid, and plasma. Twenty-eight days after MCT injection, right ventricular systolic pressure and the weight ratio of the right ventricle to the left ventricle plus septum were significantly lower in the AdEC-SOD group (42.50 +/- 1.46 mm Hg and 0.453 +/- 0.029, respectively) than in the MCT group (59.89 +/- 1.61 mm Hg and 0.636 +/- 0.022, respectively) or the Adbetagal group (61.50 +/- 2.61 mm Hg and 0.653 +/- 0.038, respectively). Moreover, vascular remodeling and proliferation of vascular smooth muscle cells in pulmonary arteries were markedly suppressed in the AdEC-SOD group. Importantly, 8-isoprostane in lung tissue was also significantly reduced in the AdEC-SOD group. EC-SOD overexpression to the lung ameliorated MCT-induced PH in rats. We suggest that EC-SOD may act as an antioxidant in PH and that increased oxidative stress may be important in the pathogenesis of MCT-induced PH.

The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity

BackgroundHuman extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.ResultsThe analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits.ConclusionThis finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

Production and Characterization of Human Extracellular Superoxide Dismutase in the Methylotrophic YeastPichia pastoris

Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.

Vascular effects of a common gene variant of extracellular superoxide dismutase in heart failure

A common gene variant of human extracellular superoxide dismutase (ecSOD), in approximately 5% of humans, is associated with increased risk of ischemic heart disease. The purpose of this study was to examine vascular effects of ecSOD with effects of the ecSOD variant (ecSOD(R213G)) in rats with heart failure. Seven weeks after coronary artery ligation, we studied rats with heart failure and sham-operated rats. Adenoviral vectors expressing human ecSOD, ecSOD(R213G), or a control virus were injected intravenously. In the aorta from rats with heart failure, responses to acetylcholine (69 +/- 4% relaxation, means +/- SE) and basal levels of nitric oxide (NO) (vasoconstrictor responses to a NO synthase inhibitor) were greatly impaired, and levels of superoxide and peroxynitrite were increased. Gene transfer of ecSOD restored responses to acetylcholine (92 +/- 2% relaxation) and basal levels of NO to normal and reduced levels of superoxide [from 2.3 +/- 0.2 to 0.9 +/- 0.2 relative light units per second per millimeter squared (RLU x s(-1) x mm(-2))] and peroxynitrite (from 2.4 +/- 0.2 to 0.9 +/- 0.1 RLU x s(-1) x mm(-2)) in the aorta from rats with heart failure. Gene transfer of ecSOD(R213G) produced little or no improvement. Responses to nitroprusside were not different among the groups. Expression of endogenous mRNA for SODs (CuZnSOD, MnSOD, and ecSOD) and endothelial NOS in the aorta was not different among the groups. In contrast to ecSOD, gene transfer of ecSOD(R213G) in rats with heart failure has minimal beneficial effect on oxidative stress, endothelial function, or basal bioavailability of NO. We speculate that greatly diminished efficacy of ecSOD(R213G) in protection against oxidative stress and endothelial dysfunction may contribute to increased risk of cardiovascular disease in humans with ecSOD(R213G).

Extracellular superoxide dismutase exists as an octamer

Human extracellular superoxide dismutase (EC-SOD) is involved in the defence against oxidative stress induced by the superoxide radical. The protein is a homotetramer stabilised by hydrophobic interactions within the N-terminal region. During the purification of EC-SOD from human aorta, we noticed that material with high affinity for heparin–Sepharose formed not only a tetramer but also an octamer. Analysis of the thermodynamic stability of the octamer suggested that the C-terminal region is involved in formation of the quaternary structure. In addition, we show that the octamer is composed of both aEC-SOD and iEC-SOD folding variants. The presence of the EC-SOD octamer with high affinity may represent a way to influence the local concentration of EC-SOD to protect tissues specifically sensitive to oxidative damage.

Gene transfer of extracellular superoxide dismutase protects against vascular dysfunction with aging

Aging is an independent risk factor for cardiovascular disease, but mechanisms leading to vascular dysfunction have not been fully elucidated. Recent studies suggest that oxidative stress may increase in blood vessels during aging. Levels of superoxide are influenced by the activity of SODs. The goal of this study was to examine the effect of extracellular superoxide dismutase (ECSOD) on superoxide levels and vascular function in an animal model of aging. Aortas from young (4-8 mo old) and old (29-31 mo old) Fischer 344 rats were examined in vitro. Relaxation of aorta to ACh was impaired in old rats compared with young rats; e.g., 3 muM ACh produced 57 +/- 4% (mean +/- SE) and 84 +/- 2% relaxation in old and young rats, respectively (P < 0.0001). Three days after gene transfer of adenovirus expressing human ECSOD (AdECSOD), the response to ACh was not affected in young rats but was improved in old rats. There was no difference in relaxation to the endothelium-independent dilator sodium nitroprusside between young, aged, and AdECSOD-treated old rats. Superoxide levels (lucigenin-enhanced chemiluminescence) were significantly increased in aged rats compared with young rats. After gene transfer of ECSOD to aged rats, superoxide levels in aorta were similar in old and young rats. Gene transfer of an ECSOD with the heparin-binding domain deleted had no effect on vascular function or superoxide levels in old rats. These results suggest that 1) vascular dysfunction associated with aging is mediated in part by increased levels of superoxide, 2) gene transfer of ECSOD reduces vascular superoxide and dysfunction in old rats, and 3) beneficial effects of ECSOD in old rats require the heparin-binding domain of ECSOD.

Delivery of antioxidative enzyme genes protects against ischemia/reperfusion–induced liver injury in mice

Hepatic ischemia/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether antioxidative gene delivery by our polylipid nanoparticles (PLNP) is an effective approach for prevention of the injury. Polyplexes of extracellular superoxide dismutase (EC-SOD) and/or catalase genes were injected via the portal vein 1 day prior to a warm I/R procedure in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. PLNP-mediated antioxidative gene delivery led to a marked increase in human EC-SOD and catalase gene expression in the liver. Liver superoxide dismutase (SOD) and catalase activity both increased approximately 10-fold. Increased liver superoxide anion levels caused by the I/R procedure were reduced to normal levels by EC-SOD gene delivery. The overexpression of these 2 antioxidative genes significantly suppressed the I/R-induced elevation of serum alanine aminotransferase (ALT) levels, decreased liver malondialdehyde content, restored glutathione reserve, and improved liver histology. In conclusion, EC-SOD or catalase gene delivery by PLNP resulted in high levels of the transgene activity in the liver, and markedly attenuated hepatic I/R injury. The protection is directly associated with elevated antioxidative enzyme activity as the result of the gene delivery. This novel approach may become a potential therapy to improve graft function and survival after liver transplantation.

1082. Delivery of Anti-Oxidative Enzyme Genes Protects Against Ischemia/Reperfusion-Induced Liver Injury in Mice

BACKGROUND: Hepatic ischemic/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether anti-oxidative gene delivery by our polycationic liposomes will be an effective approach for prevention of the injury. METHODS: Polycationic liposome-mediated extracellular superoxide dismutase (EC-SOD) or/and catalase gene delivery by portal vein injection was undertaken one day prior to warm I/R injury in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. RESULTS: Polycationic liposome-mediated anti-oxidative gene delivery led to marked hepatic transgene expression. There was a 56-fold increase in human EC-SOD gene expression in the liver 30 hours after injection of EC-SOD lipoplexes as shown by real time RT-PCR; Western blot analysis demonstrated a 10-fold increase in catalase protein levels in the liver as compared to controls. Liver SOD and catalase activity increased 16.6-fold and 9-fold, respectively. The overexpression of these two anti-oxidative genes significantly suppressed the subsequent I/R-induced acute liver injury as reflected by markedreduction of serum ALT levels (520±55 and 643±24 vs. 1217±193 units/ml, p<0.01). The I/R procedure significantly increased liver superoxide anions (699±83 vs. 20±13 RLU/sec/mg, p<0.01) as detected by a chemoluminescent method with a superoxide tracer, MCLA. These levels were markedly reduced to a nearly normal level (24±7 RLU/sec/mg) by the prior delivery of the EC-SOD gene. In addition, there were reduced liver malondialdehyde levels, and restored glutathione levels, in mice receiving either EC-SOD or catalase gene delivery. Moreover, the enhanced activation of nuclear factor-κB and AP-1 in hepatic I/R injury was minimized by the anti-oxidative gene delivery. The control lipoplexes did not affect the extent of liver injury and oxidant stress levels in this I/R model. The combination of EC-SOD and catalase lipoplex injection (in half amount of each plasmid) resulted in a slight improvement in hepatic I/R injury when compared to either alone. CONCLUSIONS: The findings demonstrate that polycationic liposome-mediated EC-SOD or catalase gene delivery led to high levels of the transgene activity in the liver, and markedly attenuated liver I/R injury. The protection is associated with enhanced anti-oxidative effects in the liver. To our knowledge, this is the first successful demonstration that polycationic liposome-mediated EC-SOD and/or catalase gene delivery ameliorates warm I/R-associated liver injury in mice. This approach may become a potential novel therapy to improve graft function and survival after liver transplantation.

Inflammatory Cells as a Source of Airspace Extracellular Superoxide Dismutase after Pulmonary Injury

Extracellular superoxide dismutase (EC-SOD) is an antioxidant abundant in the lung. Previous studies demonstrated depletion of lung parenchymal EC-SOD in mouse models of interstitial lung disease coinciding with an accumulation of EC-SOD in airspaces. EC-SOD sticks to the matrix by a proteolytically sensitive heparin-binding domain; therefore, we hypothesized that interstitial inflammation and matrix remodeling contribute to proteolytic redistribution of EC-SOD from lung parenchyma into the airspaces. To determine if inflammation limited to airspaces leads to EC-SOD redistribution, we examined a bacterial pneumonia model. This model led to increases in airspace polymorphonuclear leukocytes staining strongly for EC-SOD. EC-SOD accumulated in airspaces at 24 h without depletion of EC-SOD from lung parenchyma. This led us to hypothesize that airspace EC-SOD was released from inflammatory cells and was not a redistribution of matrix EC-SOD. To test this hypothesis, transgenic mice with lung-specific expression of human EC-SOD were treated with asbestos or bleomycin to initiate an interstitial lung injury. In these studies, EC-SOD accumulating in airspaces was entirely the mouse isoform, demonstrating an extrapulmonary source (inflammatory cells) for this EC-SOD. We also demonstrate that EC-SOD knockout mice possess greater lung inflammation in response to bleomycin and bacteria when compared with wild types. We conclude that the source of accumulating EC-SOD in airspaces in interstitial lung disease is inflammatory cells and not the lung and that interstitial processes such as those found in pulmonary fibrosis are required to remove EC-SOD from lung matrix.

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.

Extracellular superoxide dismutase (EC-SOD) gene mutations screening in a sample of Mediterranean population

The main role of superoxide dismutases (SODs) is to eliminate reactive oxygen species in cells and tissues. Extracellular SOD (EC-SOD/SOD3) is a major superoxide scavenger and it is located on cell surfaces and primarily in extracellular matrix, and binds heparan sulfates by its carboxyterminal portion. Human EC-SOD gene is located on chromosome 4 and comprises three exons and two introns. The SOD3 coding sequence is entirely located within exon 3 and has missense polymorphisms. The Arg213Gly mutation affects the function of the carboxyterminus and correlates with several diseases. In this work, we explored genetic variants within EC-SOD gene of subjects living in southern Italy. Four new variations were detected: one was silent mutation, while three were missense variations that give rise to amino acid substitutions at position 131 (F > C), 160 (V > L) and 202 (R > L) in the mature product. The Arg213Gly variant was not found. The missense mutations in the DNA of assayed 2400 chromosomes had frequencies of 5.34% for the F131C variation, 0.25% for the V160L variation and 0.84% for the R202L variation. The effect of these alterations on the metabolic activity and diseases remains to be further explained.

The Structure of Rabbit Extracellular Superoxide Dismutase Differs from the Human Protein

The cDNA sequence encoding rabbit, mouse, and rat extracellular superoxide dismutase (EC-SOD) predicts that the protein contains five cysteine residues. Human EC-SOD contains an additional cysteine residue and folds into two forms with distinct disulfide bridge patterns. One form is enzymatically active (aEC-SOD), while the other is inactive (iEC-SOD). Due to the lack of the additional cysteine residue rabbit, mouse, and rat EC-SOD are unable to generate an inactive fold identical to human iEC-SOD. The amino acid sequences predict the formation of aEC-SOD only, but other folding variants cannot be ruled out based on the heterogeneity observed for human EC-SOD. To test this, we purified EC-SOD from rabbit plasma and determined the disulfide bridge pattern. The results revealed that the disulfide bridges are homogeneous and identical to human aEC-SOD. Four cysteine residues are involved in two intra-disulfide bonds while the C-terminal cysteine residue forms an intersubunit disulfide bond. No evidence for other folding variants was detected. These findings show that rabbit EC-SOD exists as an enzymatically active form only. The absence of iEC-SOD in rabbits suggests that the structure and aspects of the physiological function of EC-SOD differs significantly between rabbit and humans. This is an important notion to take when using these animals as model systems for oxidative stress.

Gene Transfer of Extracellular Superoxide Dismutase Failed to Prevent Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

We examined the therapeutic effect of human extracellular superoxide dismutase (ECSOD) gene transfer in the prevention of delayed cerebral vasospasm after experimental subarachnoid hemorrhage (SAH) because it was reported ECSOD relieved early-stage vasospasm. Twenty mongrel dogs were divided randomly into 4 groups to serve as control, SAH, SAH+adenovirus ECSOD (AdECSOD), and SAH+no transgene (AdBglII) groups, respectively. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on day 0 and day 2. Angiography was performed at day 0 and day 7. Clinical behavior, cerebrospinal fluid (CSF) ECSOD activity, CSF leukocyte count, morphology, and human ECSOD expression (RT-PCR) in the basilar arteries were evaluated. Severe vasospasm was obtained in SAH, SAH+AdECSOD, and SAH+AdBglII gene-transferred dogs, and the residual diameters of the basilar artery were 41+/-1%, 39+/-4%, and 49+/-4%, respectively. Increased CSF activity of ECSOD was obtained in SAH+AdECSOD (162+/-23 U/mL) when compared with SAH (26+/-2) and SAH+AdBglII (25+/-3) dogs. RT-PCR confirmed successful gene transfer in the basilar arteries from SAH+AdECSOD dogs. Increased leukocyte counts were observed in the CSF and in the subarachnoid space, especially in SAH+AdECSOD and SAH+AdBglII dogs. Gene transfer of human ECSOD failed to prevent delayed cerebral vasospasm.

677. Polycationic Liposome-Mediated Extracellular Superoxide Dismutase Gene Delivery Leads to High Levels of the Transgene Expression and Prevents Acute Liver Injury in Mice

BACKGROUND: We have developed a formulation of polycationic liposomes that has been shown to be non-toxic, highly stable in the bloodsteam, and very effective in liver gene transfer in mice (Gene Therapy 2003; 10:180–187). Extracellular superoxide dismutase (EC-SOD) inhibits the generation of superoxide anions in the interstitial space of tissues, and may play a role in minimizing oxidative stress in liver injury. The aim of the present study is to investigate whether our polycationic liposome-mediated human EC-SOD gene delivery will protect against lipopolysaccharide (LPS)-induced liver toxicity in D-galactosamine (GalN)-sensitized mice. METHODS: Polycationic liposomes were generated from polycationic lipid (PCL) and cholesterol (Chol). Lipoplexes were formed by complexing liposomes with control plasmid (pEGFP-C1) or EC-SOD plasmid (pEGFP-C1-ECSOD) before use. Mice were injected with thyroid hormone (T3, 4 mg/kg, s.c.) two days before lipoplexes were injected via the portal vein. One day following lipoplex injection, mice were treated with GalN (500 mg/kg, i.p.) plus LPS (25 mg/kg, i.p.). Serum alanine aminotransferase (ALT), SOD activity, liver histology, glutathione (GSH) content and lipid peroxidation were evaluated one day after GalN/LPS exposure. Human EC-SOD gene expression in mouse liver tissue was determined by quantitative RT-PCR two days after lipoplex injection. RESULTS: somes and the control lipoplexes were stable in their size for three months at 4°C. Injection of T3, PCL-Chol liposomes or control lipoplexes did not cause a significant change in serum ALT levels. Real time quantitative RT-PCR analysis showed that human EC-SOD mRNA levels in mouse liver tissue in EC-SOD lipoplex-injected group were 55-fold higher than saline, liposome or control lipoplex controls when mouse -actin was employed as a house-keeping control gene. Serum ALT levels in animals receiving portal vein injections of EC-SOD lipoplexes plus GalN/LPS exposure were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes plus GalN/LPS exposure (62583 vs. 150865, 153194, 1880520 units/ml, p<0.01). Liver histology showed much less hepatocyte death in EC-SOD lipoplex-treated group than liposome- or control lipoplex-treated groups. Serum SOD activity in animals receiving injection of EC-SOD lipoplexes were higher than in those treated with liposomes only, or control lipoplexes (28.8 3.3 vs. 15.2 0.9, 17.4 3.4 units/ml, p<0.05). Liver GSH content in animals receiving EC-SOD lipoplex injection was preserved with less lipid peroxidation, as indicated by a lower level of malondialdehyde content. CONCLUSIONS: liposome-mediated EC-SOD gene delivery leads to the marked transgene expression, and attenuates acute liver injury due to reduced oxidative stress in GalN/LPS-exposed mice. To our knowledge, this is a first report which demonstrates that polycationic liposome-mediated EC-SOD gene delivery to the liver represents a potential therapy for hepatotoxin-induced liver injury.

Pre-emptive gene therapy using recombinant adeno-associated virus delivery of extracellular superoxide dismutase protects heart against ischemic reperfusion injury, improves ventricular function and prolongs survival.

In high-risk patients, the ideal cardiovascular gene therapy requires a strategy that provides long-term protection of myocardium against episodes of ischemic/reperfusion injury. We report the development of an efficient, long-lasting pre-emptive gene therapy strategy in a rat model of ischemic-reperfusion (I/R) injury of heart. At 6 weeks prior to myocardial injury, the human extracellular superoxide dismutase (Ec-SOD) gene was delivered by direct intramyocardial injections, using a recombinant adeno-associated virus vector. Significant myocardial protection was documented by the decrease in infarct size at 24 h post I/R, improved left ventricular function at 7 weeks postinjury, and enhanced long-term survival in the SOD treated group. This concept of preinjury delivery and 'pre-emptive' gene therapy via the expression of a secreted protein that renders paracrine therapeutic action can be an effective strategy for organ protection against future injury.

Extracellular superoxide enhances 5-HT-induced murine pulmonary artery vasoconstriction.

Accumulating evidence suggests that changes in both 5-hydroxytryptamine (5-HT) receptor activity and in the levels of reactive oxygen species (ROS) play an important role in regulating pulmonary artery (PA) vascular responsiveness, particularly in the setting of pulmonary hypertension. Therefore, we hypothesized that increased levels of superoxide enhance 5-HT-induced PA constriction. With the use of a small-vessel bioassay, 5-HT (0.01-10 microM) induced a concentration-dependent vasoconstriction in isolated wild-type murine intrapulmonary arteries (100-150 microm diameter) that was enhanced by both removal of the endothelium and by treatment with either N(G)-nitro-L-arginine methyl ester (30 microM) or xanthine (10 microM) + xanthine oxidase (0.005 U/ml). PA isolated from extracellular superoxide dismutase (EC-SOD) knockout mice also showed enhanced constriction. On the other hand, PA constriction to 5-HT was attenuated by either the addition of GR-127935 (0.1 microM, a selective inhibitor of 5-HT(1B/1D) receptor) or copper/zinc-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) and in PA isolated from transgenic mice overexpressing human EC-SOD. With the use of both oxidative fluorescent confocal microscopy and lucigenin-enhanced chemiluminescence, superoxide levels were increased significantly after 5-HT-induced PA vasoconstriction. This increase in superoxide levels could be blocked by the exogenous addition of Cu/Zn SOD (150 U/ml) or by apocynin (30 microM, an inhibitor of NADPH oxidase) but was not affected by gp91(phox) knockout mice. Overall, our results are consistent with 5-HT increasing vascular smooth muscle superoxide production via an NADPH oxidase pathway that is independent of gp91(phox), which leads to increases in extracellular superoxide levels, which in turn enhances 5-HT-induced murine pulmonary vasoconstriction.

Liposome-mediated extracellular superoxide dismutase gene delivery protects against acute liver injury in mice.

Our previous study demonstrated that polycationic liposomes are highly stable in the bloodstream and represent an effective agent for liver gene delivery. We report here that liposome-mediated extracellular superoxide dismutase (EC-SOD) gene delivery successfully prevented acute liver injury in mice. The therapeutic efficacy of EC-SOD gene delivery by polycationic liposomes was determined against the toxicity of superoxide anions and hydroxyethyl radicals in HepG2 cells and in a mouse model of acute liver injury caused by D-galactosamine and lipopolysaccharide intoxication. Transfection of HepG2 cells with an EC-SOD plasmid led to a striking increase in superoxide dismutase activity in the medium. The transfected cells had much less cell death after reactive oxygen species exposure compared with untransfected or control plasmid-transfected cells. In a model of acute liver injury, serum alanine aminotransferase levels in mice receiving portal vein injections of EC-SOD lipoplexes were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes. Liver histology confirmed that there was less cell death in the EC-SOD lipoplex-treated group. Quantitative reverse transcriptase polymerase chain reaction showed a 55-fold increase in human EC-SOD gene expression in the liver of mice injected with EC-SOD lipoplexes. Serum superoxide dismutase activity in EC-SOD lipoplex-treated mice was higher than in the control groups; this was associated with higher liver glutathione levels and reduced lipid peroxidation. In conclusion, polycationic liposome-mediated EC-SOD gene delivery protects against reactive oxygen species toxicity in vitro and against lipopolysaccharide-induced acute liver injury in D-galactosamine-sensitized mice.

The dual nature of human extracellular superoxide dismutase: one sequence and two structures.

Human extracellular superoxide dismutase (EC-SOD; EC 1.15.1.1) is a scavenger of superoxide anions in the extracellular space. The amino acid sequence is homologous to the intracellular counterpart, Cu/Zn superoxide dismutase (Cu/Zn-SOD), apart from N- and C-terminal extensions. Cu/Zn-SOD is a homodimer containing four cysteine residues within each subunit, and EC-SOD is a tetramer composed of two disulfide-bonded dimers in which each subunit contains six cysteines. The amino acid sequences of all EC-SOD subunits are identical. It is known that Cys-219 is involved in an interchain disulfide. To account for the remaining five cysteine residues we purified human EC-SOD and determined the disulfide bridge pattern. The results show that human EC-SOD exists in two forms, each with a unique disulfide bridge pattern. One form (active EC-SOD) is enzymatically active and contains a disulfide bridge pattern similar to Cu/Zn-SOD. The other form (inactive EC-SOD) has a different disulfide bridge pattern and is enzymatically inactive. The EC-SOD polypeptide chain apparently folds in two different ways, most likely resulting in different three-dimensional structures. Our study shows that one gene may produce proteins with different disulfide bridge arrangements and, thus, by definition, different primary structures. This observation adds another dimension to the functional annotation of the proteome.

Gene transfer of extracellular superoxide dismutase reduces cerebral vasospasm after subarachnoid hemorrhage.

Superoxide may play an important role in cerebral vasospasm after subarachnoid hemorrhage (SAH). Our first goal was to determine the effect of gene transfer of extracellular superoxide dismutase (ECSOD) on vasospasm after experimental SAH. Our second goal was to determine whether tissue binding of ECSOD via the heparin-binding domain (HBD) is important for the effect of the enzyme. Thus, we examined effects of gene transfer of ECSOD with the HBD deleted (ECSODDeltaHBD). Adenovirus expressing human ECSOD (AdECSOD), ECSODDeltaHBD (AdECSODDeltaHBD), or no transgene (AdBglII) was injected into the cisterna magna of anesthetized rabbits 30 minutes after induction of experimental SAH. Cerebral angiography, an assay for ECSOD activity in cerebrospinal fluid (CSF), and Western blotting for human ECSOD in the basilar artery were performed. Baseline diameter of the basilar artery averaged 0.77+/-0.01 mm (mean+/-SEM) and was similar in all treatment groups. Decreases in diameter of the basilar artery 2 days after SAH were smaller after AdECSOD (11+/-3%; n=10) than after AdBglII (25+/-4%; n=7; P<0.05). ECSOD activity was not detected in CSF before SAH and gene transfer. Of 8 rabbits treated with AdECSOD, in which ECSOD activity in CSF was measured after SAH, 5 animals had detectable ECSOD activity in CSF (46+/-13 U/mL). In these 5 rabbits, the diameter decreased by only 6+/-3%, and ECSOD protein was detected in the basilar artery. After AdECSODDeltaHBD (n=4), despite high levels of ECSOD activity in CSF (91+/-19 U/mL), vessel diameter decreased by 20+/-2%, and no ECSODDeltaHBD protein was detected in the basilar artery. Gene transfer of ECSOD reduces cerebral vasospasm after experimental SAH. Tissue binding of the enzyme is essential for cerebral vascular effects of ECSOD.