Extracellular Superoxide Dismutase Gene Research Articles

A SIMPLE ANIMAL MODEL OF EXERCISE REVEALS A MOLECULAR DETERMINANT OF LONG TERM HEALTH MAINTENANCE

Abstract At all stages of life, both dynamic gene expression changes and events that “lock in” particular programs that promote health and maintenance are critical factors in aging trajectories. We have a long-term interest in the fundamental biology of healthy maintenance, a topic that has led us to consider multiple facets of healthspan. A powerful whole-organism intervention with maintenance-promoting, anti-disease, anti-aging impact is exercise. The molecular and cellular mechanisms that mediate long-term system-wide exercise benefits, however, remain poorly understood, especially as applies to “off target” tissues that do not participate directly in training activity. We are investigating the basic biology of exercise benefits using the simple 959-celled model C. elegans. We found that multiple daily swim sessions are essential for exercise adaptation, leading to enhanced expression of muscle structural genes and improved locomotory performance. Importantly, swim exercise training enhances whole-animal health parameters such as mitochondrial respiration and mid-life survival, increases functional healthspan of pharynx and intestine, and enhances nervous system health by increasing learning ability of adults and protecting against neurodegeneration in models of tauopathy, Alzheimer’s disease, and Huntington’s disease. Remarkably, swim training only during early adulthood induces long-lasting systemic benefits that in several cases are still detectable well into mid-life. Our investigation of the molecular requirements for long-term maintenance (“legacy effects”) revealed that deletion of the sole C. elegans extracellular superoxide dismutase gene SOD-4 changes this pattern. sod-4 mutants are able to swim train to gain measurable benefits, but do not maintain the healthspan improvements that wild type animals do, defining extracellular SOD-4 as a powerful mid-life maintenance factor associated with exercise experience. Our talk will discuss transcriptomic analysis of exercise and SOD-4, as well as our current understanding of the molecular mechanisms operative. Notably, mammalian extracellular SOD ecSOD has been shown to promote exercise associated health and protection against oxidative stress insults (PMID: 32220789 Yan, Spaulding 2020), implicating ecSOD in a conserved role for health maintenance and underscoring potential for therapeutic translation.

The Effect of Extracellular Superoxide Dismutase (SOD3) Gene in Lung Cancer.

BackgroundThe recognition of new diagnostic and prognostic biological markers for lung cancer, the most severe malignant tumor, is an essential and eager study. In a microenvironment, superoxide dismutase 3 (SOD3) can adjust active oxygen, and it refers to a secreted antioxidant enzyme. It was also found to be cancer-related, and in lung cancer, it was remarkably down-regulated. More and more new cancer research focuses on the function of SOD3. Despite this, there is no good description of SOD3 function in the LC progression.MethodsThrough bioinformatics analysis, we found that SOD3 was a possible novel lung cancer gene in this study. We analyzed data sets from Gene Expression Comprehensive Database (GEO) and the Cancer Genome Atlas (TCGA), and SOD3 expression was studied in lung cancer. This study estimated the SOD3 diagnosis and prognosis through gene expression differential display, gene set enrichment analysis (GSEA), enrichment and genomes (KEGG) analysis, and gene ontology (GO). Then in order to investigate the SOD3 presentation in lung cancer cells, we used Western Blot and also applied Flow cytometry to detect the impact of anti-tumor medicine on tumor cell apoptosis.ResultsWe found that the expression level of SOD3 in lung cancer was low (P = 4.218E-29), while the survival of lung cancer patients with high SOD3 expression was shorter (LUSC p =0.00086, LUAD p=0.00038). According to the result of western blot, the expression of SOD3 in tumor cells was higher than that in normal cells. The ratio of early apoptosis induced by anti-cancer drugs was 10.5% in normal cells, 35.1% in squamous cell carcinoma and 36.9% in adenocarcinoma.The SOD3 high expression was associated with poor survival probability by multivariate analysis (HR: 1.006, 95% CI 1.002–1.011, p=0.006). Moreover, SOD3 high expression group had higher ESTIMATE scores, and larger amount of immune infiltrating cells. SOD3 expression is correlated with PDCD1 and CTLA4 expression.ConclusionsSOD3 gene can be used as a prognostic gene in lung cancer patients, and lung cancer patients with high expression of this gene can reap worse prognostic outcome. It can be used as a new clinical method and prognosticator for lung cancer patients.

The Role of Extracellular Superoxide Dismutase (Ec-SOD) Gene Polymorphism in Motor Neuron Damage of Type 2 Diabetic Peripheral Neuropathy Patients of Babylon Province-Iraq

Background: Extracellular superoxide dismutase (EC-SOD) gene polymorphism play an essential role in the progression of diabetic complications. Reactive oxygen species (ROS) were known to damage neurons by enhancing nerve lipid peroxidation, the damaging mitochondrial DNA, break-down the respiratory chain, and the cross-linking of neuron proteins. Aim: the aim of this study was to investigate the relationship between (Ec-SOD) gene polymorphism and the pathogenesis of diabetic peripheral neuropathy in type2 diabetic patients. Arg(213)Gly polymorphism of Ec-SOD gene polymorphism were studied in type2 diabetic patients with (n=30) and without DPN (n=30). Results: Polymerase chain reaction (PCR) technique were used for detection Ec-SOD polymorphisms. This technique included the use of PCR primers (Forward and Reverse) to produce a restriction site in the amplified EC-SOD gene product just with the polymorphic base. Then, the product of (PCR) was digested with Eco52I restriction enzyme to detect Arg(-213)Gly polymorphic position. The results of Arg(-213)Gly polymorphism showed that the frequency of Arg/Arg, Arg/Gly, and Gly/Gly were 70%, 13.3%, and 16.6% in healthy control subject and 53.3%, 16.6%, and 30% in diabetic without neuropathy countered by 23.3%, 26.6%, and 50% in diabetic with neuropathy. This proposed that the Arg (-213)Gly polymorphism in the EcSOD gene is significantly associated with a risk for progression of diabetic peripheral neuropathy. Conclusions: Arg(-213)Gly polymorphism of Ec-SOD gene was associated with diabetic peripheral neuropathy in type2 diabetic patients of Babylon Province

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Association of extracellular superoxide dismutase gene methylation with cerebral infarction

To assess the association of extracellular superoxide dismutase (EC-SOD) gene methylation with cerebral infarction. Eighty-three patients with cerebral infarction and 94 healthy controls were enrolled. Based on cerebral MR findings, the size of infarction, extent of intracranial atherosclerosis, the National Institutes of Health Stroke Scale (NIHSS) score, and Barthel index were calculated. Methylation-specific PCR was used to analyze the methylation status of the EC-SOD gene in peripheral blood samples and its correlation with cerebral infarction. The rate of EC-SOD gene promoter region methylation of the cerebral infarction group was lower than that of the control group (30.1% vs. 53.2%, P < 0.05). Patients with larger area of cerebral infarction (>4 cm in diameter) showed a lower methylation rate than those with a smaller cerebral infarction (0 vs. 39.1%, P < 0.05). Based on their cerebral MRA, 57 patients were divided into none, mild, moderate, and severe cerebral arteriosclerosis groups. The rate of EC-SOD gene methylation of the four groups showed a downward trend (at 45.5%, 42.9%, 23.8%, and 14.3%, respectively), though no statistical significance was found (P > 0.05). For the cerebral infarction group, those with higher rate of methylation had lower NIHSS scores (P < 0.05) but insignificantly higher Barthel index (P > 0.05). The EC-SOD methylation frequency of case group was lower than the control group. The methylation status is associated with the size of cerebral infarction, degree of cerebral arteriosclerosis and severity of neurological impairment.

Correlation between Helicobacter pylori infection and polymorphism of adiponectin gene promoter -11391G/A, superoxide dismutase gene in nonalcoholic fatty liver disease.

To investigate the correlation between Helicobacter pylori (H. Pylori) infection and polymorphism of adiponectin gene promoter -11391G/A, extracellular superoxide dismutase (EC-SOD) gene in nonalcoholic fatty liver disease (NAFLD). From June, 2010 to July, 2014, a hospital-based 1:1 matched case-control study was carried out, with 600 cases of NAFLD and 600 healthy people in the First Affiliated Hospital of Xinxiang Medical University. The genetic polymorphisms of adiponectin gene promoter -11391G/A and EC-SOD were analyzed by polymorphism-polymerase chain reaction (PCR) technique in peripheral blood leukocytes of the subjects. 14C-urea breath test (14C-UBT) was used to test 14C disntegration per minute (DPM) for evaluating the infections status of H. Pylori. The synergistic effect between the two mutants and the gene-environment interaction of the genotypes with H. Pylori infection were analyzed. The frequencies of -11391G/A (AA) and EC-SOD (CG+GG) were 50.67% and 50.33% in NAFLD cases, 23.83% and 24.17% in healthy controls, respectively. Statistical tests showed significantly higher frequencies of -11391G/A (AA) and EC-SOD (CG+GG) in the NAFLD group (-11391G/A: P=0.0051; EC-SOD: P=0.0057). The risk of NAFLD with -11391G/A (AA) was significantly higher than those with -11391G/A(GG+GA) (OR=3.2822, 95% CI 1.9170 to 5.2039). The individuals who carried EC-SOD (CG+GG) had a high risk of NAFLD (OR=3.1800, 95% CI 1.7974 to 5.2391). Combined analysis of the polymorphisms showed that percentage of -11391G/A (AA)/EC-SOD (CG+GG) in the NAFLD group was significantly higher than that in the control groups (25.50% vs 5.83%, P=0.0039). The people who carried with -11391G/A (AA)/EC-SOD (CG+GG) had a high risk of NAFLD (OR=10.3190, 95% CI 8.1869 to 20.5102). The H. Pylori infection rate in the NAFLD group was significantly higher than that in the control group (OR=3.1667, 95% CI 1.9139 to 5.7443, P=0.0062), and statistical analysis suggested a positive correlation between H. Pylori infection and NAFLD with -11391G/A (AA) and EC-SOD (CG+GG) (-11391G/A: γ=1.8532; EC-SOD: γ=1.7899). These carriers of -11391G/A(AA) and EC-SOD (CG+GG) genotypes may have a high risk of NAFLD, and the gene genotypes can interact with H. Pylori infection in the pathogenesis of NAFLD. Therefore, effective prevention measures for NAFLD should consider eradicating H. Pylori or regulating gene expression.

English

Free radical-induced damage plays a significant role in the development of type 2 diabetes and its complications. Extracellular superoxide dismutase (EC-SOD) or SOD3 is one of the important scavenger enzymes that protects against oxidative damage and the formation of free radicals. EC-SOD is a copper- and zinc-containing secretory enzyme located in tissues and primarily in the extracellular matrix. EC-SOD has been proposed to protect against pancreatic beta-cell damage, insulin resistance and vascular function in diabetic patients. The association between EC-SOD gene polymorphism and Type 2 diabetes mellitus is not completely understood. The aim of the study is to investigate the EC-SOD gene polymorphism in the exon 3 region and the susceptibility to type 2 diabetes mellitus and its complications among Lebanese patients. For this purpose, a genotype analysis involving 60 Type 2 Diabetes patients and 46 control subjects was carried out using PCR-based direct DNA sequencing. Results showed that the frequency of the Ala40Thr polymorphism of EC-SOD gene was significantly higher in type 2 diabetic patients compared to control group. Evaluation of clinical parameters of diabetic patients with their relative genotypes showed that patients with the Thr allele have higher HbA1c levels and fasting blood glucose compared to their Ala genotypes indicating a poor diabetes control in these patients. When diabetic complications including retinopathy, nephropathy and cardiovascular complications were correlated with Ala40Val polymorphism, a significant association was detected for complications and particularly for cardiovascular diseases in diabetic patients. Key words: Type 2 diabetes mellitus, oxidative stress, extracellular superoxide dismutase, genetic biomarker.

Genetic polymorphisms in extracellular superoxide dismutase Leu53Leu, Arg213Gly, and Ala40Thr and susceptibility to type 2 diabetes mellitus.

The most common type of endocrine disease is type 2 diabetes mellitus (T2DM); genetic factors contribute to the development to T2DM. In this study, we investigated the role of the Leu53Leu, Arg213Gly, and Ala40Thr polymorphisms in extracellular superoxide dismutase (EC-SOD) gene in the development of T2DM in a Chinese population. DNA was extracted from peripheral blood samples obtained from 256 T2DM patients and 324 control subjects recruited from our hospital between January 2013 and March 2015. DNA was genotyped by polymerase chain reaction-restriction fragment length polymorphism. The obtained data was then statistically analyzed. The chi-square test revealed a statistically significant difference in the genotype frequencies of EC-SOD Ala40Thr (χ2 = 13.26, P = 0.001) between the patients and controls. Unconditional regression analysis indicated that the GA and AA genotypes of EC-SOD Ala40Thr were associated with an increased risk of T2DM compared to the GG genotype {adjusted odds ratio (OR) [95% confidence interval (CI)] = 1.46 (1.01-2.11) and 2.67 (1.48-4.85), respectively}. In the dominant model, the GA+AA genotype of EC-SOD Ala40Thr was correlated with a higher risk of T2DM, in comparison with the GG genotype (OR = 1.64, 95%CI = 1.16-2.33). In the recessive model, AA of EC-SOD Ala40Thr showed a 2.19-fold higher risk of developing T2DM than the GG+GA genotype. In conclusion, people with the Ala40Thr polymorphism in EC-SOD are at a higher risk of developing T2DM; therefore, this may be utilized as a biomarker for early screening of T2DM in a Chinese population.

Extracellular Superoxide Dismutase: Growth Promoter or Tumor Suppressor?

Extracellular superoxide dismutase (SOD3) gene transfer to tissue damage results in increased healing, increased cell proliferation, decreased apoptosis, and decreased inflammatory cell infiltration. At molecular level, in vivo SOD3 overexpression reduces superoxide anion (O2 −) concentration and increases mitogen kinase activation suggesting that SOD3 could have life-supporting characteristics. The hypothesis is further strengthened by the observations showing significantly increased mortality in conditional knockout mice. However, in cancer SOD3 has been shown to either increase or decrease cell proliferation and survival depending on the model system used, indicating that SOD3-derived growth mechanisms are not completely understood. In this paper, the author reviews the main discoveries in SOD3-dependent growth regulation and signal transduction.

Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation.

Exendin-4 is an agonist of the glucagon-like peptide 1 receptor (GLP-1R) and is used in the treatment of type 2 diabetes. Since human GLP-1R has been identified in various cells besides pancreatic cells, exendin-4 is expected to exert extrapancreatic actions. It has also been suggested to affect gene expression through epigenetic regulation, such as DNA methylation and/or histone modifications. Furthermore, the expression of extracellular-superoxide dismutase (EC-SOD), a major SOD isozyme that is crucially involved in redox homeostasis, is regulated by epigenetic factors. In the present study, we demonstrated that exendin-4 induced the demethylation of DNA in A549 cells, which, in turn, affected the expression of EC-SOD. Our results showed that the treatment with exendin-4 up-regulated the expression of EC-SOD through GLP-1R and demethylated some methyl-CpG sites (methylated cytosine at 5'-CG-3') in the EC-SOD gene. Moreover, the treatment with exendin-4 inactivated DNA methyltransferases (DNMTs), but did not change their expression levels. In conclusion, the results of the present study demonstrated for the first time that exendin-4 regulated the expression of EC-SOD by reducing the activity of DNMTs and demethylation of DNA within the EC-SOD promoter region in A549 cells.

Oxidized low-density lipoprotein accelerates the destabilization of extracellular-superoxide dismutase mRNA during foam cell formation

Extracellular-superoxide dismutase (EC-SOD) is one of the main anti-oxidative enzymes that protect cells against the damaging effects of superoxide. In the present study, we investigated the regulation of EC-SOD expression during the oxidized low density lipoprotein (oxLDL)-induced foam cell formation of THP-1-derived macrophages. The uptake of oxLDL into THP-1-derived macrophages was increased and EC-SOD expression was decreased in a time-dependent manner by oxLDL. Furthermore, EC-SOD suppression by oxLDL was mediated by the binding to scavenger receptors, especially CD36, from the results with siRNA experience. EC-SOD expression is known to be regulated by histone acetylation and binding of the transcription factor Sp1/3 to the EC-SOD promoter region in human cell lines. However, oxLDL did not affect these processes. On the other hand, the stability of EC-SOD mRNA was decreased by oxLDL. Moreover, oxLDL promoted destabilization of ectopically expressed mRNA from EC-SOD or chimeric Cu,Zn-SOD gene with the sequence corresponding to 3′UTR of EC-SOD mRNA, whereas oxLDL had no effect on ectopic mRNA produced from EC-SOD gene lacking the sequence. These results suggested that oxLDL decreased the expression of EC-SOD, which, in turn, accelerated the destabilization of EC-SOD mRNA, leading to weaker protection against oxidative stress and atherosclerosis.

Successful Mitigation of Radiation Injuries in Mice using Mesenchymal Stem Cells Genetically Modified to Secrete Extracellular Superoxide Dismutase

Oxidative stress is a major determinant for radiation-induced tissue injuries. We present a novel method that harnesses the power of migration of mesenchymal stem cells (MSCs) to radiation injured tissues and adenovirusmediated extracellular superoxide dismutase (ECSOD) gene therapy for oxidative stress. This report demonstrates for the first time that intravenous administration of MSCs genetically modified to secrete ECSOD at 24 hours after radiation exposure can improve survival from 10% to 52%, extend lifespan for 207 days, retard cataract formation for 39 days, and prevent carcinogenesis in mice. For proof-of-concept, we further demonstrate for the first time that human MSCs can be genetically modified with adenoviral vector to secrete high levels of biologically active ECSOD. Our findings suggest that mesenchymal stem cell-based antioxidant gene therapy has the potential for mitigation of radiation injuries in humans as a consequence of radiological and nuclear emergencies, space radiation exposure, and cancer radiotherapy toxicity.

The superoxide dismutase from red claw crayfish, Cherax quadricarinatus: molecular cloning and characterization analysis.

In the present study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of ecCuZnSOD is 498 bp and encodes a 166 amino acids (aa) protein, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein. The amino acid sequences of C. quadricarinatus ecCuZnSOD and mtMnSOD showed high similarities with those of ecCuZnSODs and mtMnSODs of other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of C. quadricarinatus were highly expressed in hepatopancreas, hemocytes, intestine, and gill; low transcript levels were seen in other tissues (heart, muscle, and nerve). The immune responses of ecCuZnSOD and mtMnSOD were studied following inoculation with Spiroplasma eriocheiris and Aeromonas hydrophila. After S. eriocheiris or A. hydrophila challenge, mRNA transcription of ecCuZnSOD and mtMnSOD in hemocytes and gill was upregulated. mRNA transcription of ecCuZnSOD in the hepatopancreas was also upregulated after S. eriocheiris or A. hydrophila inoculation. mtMnSOD in hepatopancreas was upregulated after A. hydrophila inoculation, whereas this was down-regulated after S. eriocheiris challenge. After S. eriocheiris and A. hydrophila challenge, total SOD activity and CuZnSOD activity both increased compared to control group. The results showed that these SODs from C. quadricarinatus likely play an important role in protecting some tissues from reactive oxygen intermediates produced during challenge from S. eriocheiris and A. hydrophila.

Correlations between polymorphisms of extracellular superoxide dismutase, aldehyde dehydrogenase-2 genes, as well as drinking behavior and pancreatic cancer.

To investigate the correlation between drinking behavior combined with polymorphisms of extracellular superoxide dismutase (EC-SOD) and aldehyde dehydrogenase-2 (ALDH2) genes and pancreatic cancer. The genetic polymorphisms of EC-SOD and ALDH2 were analyzed by polymerase chain reaction restriction fragment length polymorphism in the peripheral blood leukocytes obtained from 680 pancreatic cancer cases and 680 non-cancer controls. Subsequently the frequency of genotype was compared between the pancreatic cancer patients and the healthy controls.The relationship of drinking with pancreatic cancer was analyzed. The frequencies of EC-SOD (C/G) and ALDH2 variant genotypes were 37.35% and 68.82% respectively in the pancreatic cancer cases, and were significantly higher than those in the healthy controls (21.03% and 44.56%, all P<0.01). People who carried EC-SOD (C/G) (OR=2.24, 95% CI= 1.81-4.03, P<0.01) or ALDH2 variant genotypes (OR=2.75, 95% CI=1.92-4.47, P<0.01) had a high risk to develop pancreatic cancer. Those who carried EC-SOD (C/G) genotype combined with ALDH2 variant genotype had a high risk for pancreatic cancer (29.56% vs. 6.76%, OR=7.69, 95% CI=3.58-10.51, P<0.01). The drinking rate of the pancreatic cancer group (64.12%) was significantly higher than that of the control group (40.15%; OR=2.66, 95% CI=1.30-4.42, P<0.01). An interaction between drinking and EC-SOD (C/G)/ALDH2 variant genotypes increased the risk of occurrence of pancreatic cancer (OR=25.00, 95% CI= 11.87-35.64, P<0.01). EC-SOD (C/G), ALDH2 variant genotypes and drinking might be the risk factors of pancreatic cancer.

Extracellular superoxide dismutase suppresses hypoxia-inducible factor-1α in pancreatic cancer

AbstractHypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that governs cellular responses to reduced oxygen availability by mediating crucial homeostatic processes and is a major survival determinant for tumor cells growing in a low-oxygen environment. Clinically, HIF-1α seems to be important in pancreatic cancer, as HIF-1α correlates with metastatic status of the tumor. Extracellular superoxide dismutase (EcSOD) inhibits pancreatic cancer cell growth by scavenging nonmitochondrial superoxide. We hypothesized that EcSOD overexpression leads to changes in the O2−/H2O2 balance modulating the redox status affecting signal transduction pathways. Both transient and stable overexpression of EcSOD suppressed the hypoxic accumulation of HIF-1α in human pancreatic cancer cells. This suppression of HIF-1α had a strong inverse correlation with levels of EcSOD protein. Coexpression of the hydrogen peroxide-removing protein glutathione peroxidase did not prevent the EcSOD-induced suppression of HIF-1α, suggesting that the degradation of HIF-1α observed with high EcSOD overexpression is possibly due to a low steady-state level of superoxide. Hypoxic induction of vascular endothelial growth factor (VEGF) was also suppressed with increased EcSOD. Intratumoral injections of an adenoviral vector containing the EcSOD gene into preestablished pancreatic tumors suppressed both VEGF levels and tumor growth. These results demonstrate that the transcription factor HIF-1α and its important gene target VEGF can be modulated by the antioxidant enzyme EcSOD.

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409]

A bioluminescent transgenic mouse model: Real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5′ region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5′-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.

Transfection of ECSOD to Rhesus bone marrow mesenchymal stem cells in vitro

This study was aimed to investigate the biological effects of Rhesus bone marrow mesenchymal stem cells (R-BMMSC) transfected by adenovirus bearing extracellular superoxide dismutase gene (AD-ECSOD). Using density gradient centrifugation and adherent culture way, the R-BMMSC transfected by AD-ECSOD and reporter gene EGFP were isolated, cultured and purified; the transfection efficiency was detected by fluorescence microscopy and flow cytometry; the ECSOD protein expression in cell culture supernatant were detected by ELISA; the surface antigens on R-BMMSC (CD34, CD29, CD45, CD90, HLA-DR) were detected by flow cytometry; and differentiation capability of transfected R-BMMSC were detected by oil red O and alizarin staining; the proliferation capability of R-BMMSC was assay by MTT method. The results showed that the transfection efficiency of AD-ECSOD (MOI 500, 1 000, 1 500 and 2 000) for R-BMMSC was > 95%. At 24 h after transfection, the ECSOD protein could be detected in cell culture supernatant, and its level was significantly higher than that of control group (P < 0.01). At 48 h after transfection, the expression level of ECSOD protein on MOI 1 500 and 2 000 was the highest. The proliferative capability, surface antigen expression and multi directive differentiation ability of transfected R-BMMSC were similar to non-transfected R-BMMSC. It is concluded that the AD-ECSOD can effectively transfect the R-BMMSC without influences on its biological features.

Cloning and Expression of the Human Extracellular Superoxide Dismutase (EC-SOD) Gene in Probiotic Lactobacillus Casei

Cloning and Expression of the Human Extracellular Superoxide Dismutase (EC-SOD) Gene in Probiotic Lactobacillus Casei

Adeno‐associated virus serotype 9 administered systemically after reperfusion preferentially targets cardiomyocytes in the infarct border zone with pharmacodynamics suitable for the attenuation of left ventricular remodeling

Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2-3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.