Extracellular Superoxide Dismutase Transgenic Mice Research Articles

Overexpression of extracellular superoxide dismutase protects against brain injury induced by chronic hypoxia.

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of 18FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.

Loss of Extracellular Superoxide Dismutase Induces Severe IL-23-Mediated Skin Inflammation in Mice

Psoriasis is a common chronic and complex autoimmune inflammatory skin disorder. The histological characteristics of psoriasis are epidermal hyperplasia, mononuclear leukocyte infiltration into the dermis, and increased angiogenesis. However, the mechanisms involved in the pathogenesis of psoriasis remain unclear. Extracellular superoxide dismutase (EC-SOD) has antichemotactic activities. Because immune cell infiltration is seen in psoriatic lesions and psoriasis patients express low levels of EC-SOD, we hypothesized that the lack of EC-SOD induces more severe IL-23-mediated psoriasis-like skin inflammation. To test this hypothesis, we determined whether the loss of EC-SOD causes more severe IL-23-induced skin inflammation. Ear skin after IL-23 administration was thicker in EC-SOD knockout (KO) mice compared with wild-type mice. In addition, infiltration of CD4(+) T cells, macrophages, and dendritic cells (DCs) into IL-23 injection sites was more elevated in EC-SOD KO mice. The expression of proinflammatory cytokines and chemokines was also more elevated in EC-SOD KO mice, and EC-SOD KO DCs expressed a higher level of MHCII. Finally, EC-SOD transgenic mice showed much less severe IL-23-induced skin inflammation. Therefore, EC-SOD may inhibit IL-23-induced psoriasis-like inflammation through the inhibition of immune cell infiltration and immune responses. These results suggest that EC-SOD could be a possible candidate for management of psoriasis.

Enhanced expression of EcSOD in skeletal muscle blocks chronic heart failure–induced muscle atrophy and exercise intolerance in mice

We have recently identified nitric oxide (NO)‐dependent antioxidant defense system underlying the resistance of oxidative myofibers to catabolic muscle wasting. To further ascertain the function of this defense system in vivo, we subjected mice to dexamethasone (Dex) injection with/without systemic administration of NO donor S‐nitrosoglutathione (GSNO). We show that Dex induced muscle atrophy and increased E3 ligases MAFbx/atrogin‐1 mRNA along with reduction of extracellular superoxide dismutase (EcSOD) protein in glycolytic muscles, which could be prevented by GSNO. Consistent with the hypothesis that EcSOD is important in maintaining muscle mass and function against cachexia, we observed that Dex‐induced muscle atrophy could be significantly attenuated by electric pulse‐mediated gene transfer of EcSOD and in muscle‐specific EcSOD transgenic mice [muscle creatine kinase (MCK)‐EcSOD]. Importantly, when MCK‐EcSOD mice were crossbred with cardiac‐specific calsequstrin [α‐myosin heavy chain (α‐MHC)‐CSQ] mice, a genetic model of chronic heart failure, they presented significant attenuation of catabolic muscle wasting with well‐preserved whole body muscle strength and exercise tolerance. These findings strongly support the functional importance of EcSOD in protecting against cachexia‐induced muscle wasting and loss of function.

Over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC-SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.

EC-SOD induces apoptosis through COX-2 and galectin-7 in the epidermis

Extracellular superoxide dismutase (EC-SOD) is an anti-oxidant enzyme found in the extracellular matrix of tissues, and plays an important role in the prevention of many diseases caused by oxidative stress. However, other functions of EC-SOD in epidermis are not well known. We investigated the functions of EC-SOD in epidermis using keratinocyte cell line and EC-SOD transgenic mice. Expression of galectin-7 in pEC-SOD transfected cells or skin of EC-SOD transgenic mice was detected by western blot analysis. The percentage of apoptotic cells was determined by propidium iodide staining and subsequent FACS analysis. COX-2 siRNA or scrambled siRNA was transfected into HaCaT cells and western blot analysis was performed to detect pro-apoptotic protein levels. The epidermis of EC-SOD transgenic mice was thinner than wild type mice. In addition, we showed that the thin epidermis of EC-SOD transgenic mice results from the apoptosis of epidermal cells. To elucidate which molecules are involved in EC-SOD-induced apoptosis, we utilized two-dimensional electrophoresis; the results showed that the epidermis of EC-SOD transgenic mice produces more galectin-7, a pro-apoptotic factor, than the wild type. Furthermore, we showed that the transfection of EC-SOD-expressing plasmids induces the production of galectin-7, and pro-apoptotic proteins in keratinocytes. This suggests that EC-SOD induces apoptosis through increased galectin-7 expression. Finally, we demonstrated that EC-SOD-induced galectin-7 results from the production of COX-2. Our results imply that EC-SOD plays a role not only as a reactive oxygen species scavenger, but also as a pro-apoptotic factor via COX-2/galectin-7 pathways in the epidermis.

Decreased pulmonary extracellular superoxide dismutase during systemic inflammation

Oxidative damage is a major cause of lung injury during systemic inflammatory response syndrome. In this study, the expression of an antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), and its protective role against pulmonary oxidative damage were investigated using mouse models of systemic inflammation. Intraperitoneal injection with bacterial endotoxin lipopolysaccharides (LPS; 20 mg/kg) caused oxidative damage in lungs as assessed by increased tyrosine nitration in proteins. LPS administration also resulted in a rapid and significant loss of more than 80% of pulmonary EC-SOD in a time- and dose-dependent manner, but other types of SODs, cytoplasmic CuZn-SOD and mitochondrial Mn-SOD, were not affected. EC-SOD protein is most abundant in lungs but also present at high levels in other tissues such as heart and white fat; however, the LPS-mediated decrease in this enzyme was most apparent in the lungs. Intravenous injection of mice with tumor necrosis factor α (10 μg per mouse) also caused a 60% decrease in EC-SOD in the lungs, suggesting that the EC-SOD down-regulation is mediated by this LPS-inducible inflammatory cytokine. A protective role for EC-SOD against LPS-mediated systemic inflammation was shown by an increased survival rate (75% vs 29% in 5 days) and decreased pulmonary oxidative damage in EC-SOD transgenic mice that overexpress the human EC-SOD gene. These results demonstrate that the inflammation-mediated EC-SOD down-regulation has a major pathophysiological impact during the systemic inflammatory response syndrome.

EC-SOD Suppresses Contact Hypersensitivity in Mouse Skin by Impairing Langerhans Cell Migration

Extracellular superoxide dismutase (EC-SOD) is primarily a tissue enzyme and has been implicated in the modulation of inflammatory response. The biological role of EC-SOD in skin, however, has rarely been investigated. In this study, we aim to explore the effects of EC-SOD on the inflammatory response in skin by evaluating the contact hypersensitivity response (CHS) in EC-SOD transgenic mice. Transgenic mice with skin-specific expression of EC-SOD were sensitized and challenged with 2,4,6-trinitro-1-chlorobenzene (TNCB), followed by measurement of ear swelling. EC-SOD transgenic mice showed significantly reduced CHS responses compared with wild-type mice. Histological evaluation of the challenged ears of EC-SOD transgenic mice revealed diminished infiltration of inflammatory cells with a failure to induce expression of inflammatory cytokines, such as tumor necrosis factor-alpha and IFN-gamma, on sensitization and challenge with TNCB. Furthermore, Langerhans cell migration to lymph nodes was impaired in EC-SOD transgenic mice. These results indicate that EC-SOD downregulates CHS through inhibition of the inflammatory response, suggesting a possible therapeutic regimen in inflammatory skin diseases.

Inhibition of the TPA-induced cutaneous inflammation and hyperplasia by EC-SOD

This study reports the roles of extracellular superoxide dismutase (EC-SOD) in the cutaneous inflammation and hyperplasia with 12- O-tetradecanoylphorbol-3-acetate (TPA) application in EC-SOD transgenic mice (Tg EC-SOD). Topical double TPA treatment induced the various inflammatory changes including the epidermal thickness, elevated the PCNA-labeling index, the edema formation, and increased production of hydrogen peroxide (H 2O 2) in wild type mice (WT). These changes were markedly suppressed in TPA-treated Tg EC-SOD. The expressions of the inflammatory cytokines, IL-1α and IL-1β, were reduced in the TPA-treated Tg EC-SOD compared with those in TPA-treated WT. The expression of IL-1α was significantly increased in the skin of TPA-treated WT, especially in the basal and suprabasal layers, but it was restricted focally in basal layer of the skin of TPA-treated Tg EC-SOD. The number of infiltrating inflammatory cells and the IL-1β expressing cells was obviously reduced in TPA-treated Tg EC-SOD in comparison with TPA-treated WT. The result suggests that EC-SOD might play an important role in the suppression of TPA-induced cutaneous inflammation and epidermal hyperplasia by regulating the expression of IL-1α and IL-1β, although the mechanisms remain to be elucidated.

Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro

The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM+5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199+1% FBS (n=108) or (2) DMEM+5% FBS (n=112), cultured for 48h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7±3.1ng/ml) was the highest for all groups (P<0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n=84), (2) Tg-CMEF at day 2 (n=92) or (3) Tg-CMEF at day 4 (n=98), cultured for 48h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P<0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.

Aging-Dependent Alterations in Synaptic Plasticity and Memory in Mice That Overexpress Extracellular Superoxide Dismutase

Oxidative damage caused by reactive oxygen species (ROS) has been proposed to be critically involved in several pathological manifestations of aging, including cognitive dysfunction. ROS, including superoxide, are generally considered as neurotoxic molecules whose effects can be alleviated by antioxidant enzymes. However, ROS also are known to be necessary components of the signal transduction cascades underlying normal synaptic plasticity. Therefore, we reasoned that the role that ROS and antioxidant enzymes play in modulating neuronal processes varies over the lifespan of an animal. We examined hippocampal long-term potentiation (LTP) and memory-related behavioral performance in transgenic mice overexpressing extracellular superoxide dismutase (EC-SOD) and their wild-type littermates at different ages. We found that aged EC-SOD transgenic mice exhibited enhanced hippocampal LTP, better cerebellum-dependent motor learning, and better hippocampus-dependent spatial learning compared with their wild-type littermates. We also found that EC-SOD overexpression impaired contextual learning, but the impairment was decreased in the aged transgenic mice. At the molecular level, aged EC-SOD transgenic mice had lower superoxide levels, a decrease in protein carbonyl levels, and a decrease in p38 and extracellular signal-regulated kinase 2 phosphorylation compared with aged wild-type mice. Our findings suggest that elevated levels of superoxide contribute to aging-related impairments in hippocampal LTP and memory, and that these impairments can be alleviated by overexpression of EC-SOD. We conclude that there is an age-dependent alteration in the role of superoxide in modulating synaptic plasticity and learning and memory.

Overexpression of Extracellular Superoxide Dismutase (EC-SOD) in Mouse Skin Plays a Protective Role in DMBA/TPA-Induced Tumor Formation

Extracellular superoxide dismutase (EC-SOD, EC 1.15.1.1) is a major antioxidant enzyme that is located in the extracellular matrix and on the cell surface. EC-SOD protects against cell and tissue damage initiated by extracellular-produced reactive oxygen species (ROS). We investigated a major role of EC-SOD in the development of tumor formation. In this study, we reported that skin-specific overexpressed EC-SOD transgenic mice showed half the number of tumors compared with the nontransgenic mice in the dimethylbenzanthracene (DMBA)-initiated and a 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage skin carcinogenesis model. This model showed a significant increase of the epidermal cell proliferation in the nontransgenic mice, but the proliferative response in the transgenic mice was delayed. The 8-hydroxy-2'-deoxyguanosine (8OH-dG) detection assay showed that the oxidative DNA damage was significantly higher in the nontransgenic mice than in the transgenic mice after TPA treatments. Overall, EC-SOD overexpression inhibited the TPA-induced cell proliferation and DNA damage, and reduced the subsequent formation of tumors. Our data suggest that EC-SOD plays a protective role in DMBA/TPA-induced skin carcinogenesis.

Extracellular superoxide dismutase attenuates lung injury after hemorrhage.

Reperfusion of the lung after hemorrhage generates free radicals such as superoxide (O(2)(.)) that may injure the lung; however, the relative importance of intracellular versus extracellular free radicals is unclear. The superoxide dismutases (SOD) are the primary enzymatic method to reduce superoxide. We examined whether lung-specific overexpression of extracellular superoxide dismutase (EC-SOD) would attenuate hemorrhage-induced lung injury. Wild-type mice and mice overexpressing the human EC-SOD gene with a lung-specific promoter were hemorrhaged by removing 30% of blood volume. After hemorrhage, the lung wet to dry weight ratios increased from 5.4 +/- 0.11 in unmanipulated control mice to 6.3 +/- 0.16 in wild-type mice, but to only 5.60 +/- 0.17 in the EC-SOD transgenic mice (p < 0.05 compared with hemorrhaged wild-type). Hemorrhage-induced lipid peroxidation, as assessed by lung F(2) isoprostanes, was lower in the EC-SOD transgenic mice (3.4 +/- 0.3 microg/lung) compared with wild-type mice (1.9 +/- 0.2 microg/lung; p < 0.05). Compared with wild-type, EC-SOD transgenic mice had attenuated the hemorrhage-induced increase in both pulmonary nuclear factor kappa B (NK-kappaB) activation (relative absorbance 1.1 +/- 0.2 for EC-SOD transgenic versus 2.5 +/- 0.1 for wild-type; p < 0.05) and myeloperoxidase activity (5.1 +/- 0.87 units/g for EC-SOD transgenic versus 11.3 +/- 1.8 units/g for wild-type; p < 0.01). Thus, overexpression of pulmonary EC-SOD in the mouse lung attenuates lung injury after hemorrhage.

Impairment of Long-term Potentiation and Associative Memory in Mice That Overexpress Extracellular Superoxide Dismutase

Reactive oxygen species, including superoxide, generally are considered neurotoxic molecules whose effects can be alleviated by antioxidants. Different from this view, we show that scavenging of superoxide with an antioxidant enzyme is associated with deficits in hippocampal long-term potentiation (LTP), a putative neural substrate of memory, and hippocampal-mediated memory function. Using transgenic mice that overexpress extracellular superoxide dismutase (EC-SOD), a superoxide scavenger, we found that LTP was impaired in hippocampal area CA1 despite normal LTP in area CA3. The LTP impairment in area CA1 could be reversed by inhibition of EC-SOD. In addition, we found that EC-SOD transgenic mice exhibited impaired long-term memory of fear conditioning to contextual cues despite exhibiting normal short-term memory of the conditioning experience. These findings strongly suggest that superoxide, rather than being considered exclusively a neurotoxic molecule, should also be considered a signaling molecule necessary for normal neuronal function.

Cold-induced brain edema in mice. Involvement of extracellular superoxide dismutase and nitric oxide

The role of extracellular superoxide in the pathogenesis of vasogenic edema was studied using transgenic mice expressing a 5-fold increase in extracellular superoxide dismutase (EC-SOD) activity in their brains. Increased EC-SOD expression offered significant protection against edema development after cold-induced injury (44% less edema than nontransgenic littermates, p < 0.05). Since iron may contribute to vasogenic edema by catalyzing the production of hydroxyl radical from superoxide and hydrogen peroxide, the effects of the chelator deferoxamine were studied. Deferoxamine reduced edema formation after cold-induced injury (43% less edema than controls, p < 0.05); however, treatment with iron-saturated deferoxamine also reduced edema development in mice (32-48% less edema, p < 0.05). This suggested that the protection offered by deferoxamine was independent of its ability to chelate iron. An iron-independent mechanism by which superoxide can contribute to vasogenic edema is via reaction with nitric oxide to produce the potentially toxic peroxynitrite anion, which is also scavenged by deferoxamine. Mice treated with an inhibitor of nitric oxide synthase were protected against cold-induced edema (37% less edema, p < 0.05). EC-SOD transgenic mice received no additional protection by inhibition of nitric oxide synthesis, supporting this novel alternative mechanism of edema formation.