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Superoxide dismutase 3 (SOD3), an antioxidant enzyme, is known as extracellular SOD (EC-SOD) because it is the predominant form in extracellular fluids. The diversity of plasma EC-SOD concentration is associated with the SOD3 p.R231G missense variant genotype. To clarify the association among SOD3 genotype, plasma EC-SOD concentration, and comorbidity in Oldest Old, we analyzed genome-wide associations with plasma EC-SOD concentration and associations between EC-SOD concentration and medical history classified by the SOD3 genotype in the Very Old (85-99 years old, n = 505) and Centenarians (over 100 years old, n = 595). The results revealed that SOD3 p.R231G was the most significant variant associated with plasma EC-SOD concentration. Although no significant difference was observed in medical histories between the SOD3 p.R231G variant non-carriers and carriers, higher EC-SOD concentration in plasma of SOD3 p.R231G variant non-carriers was associated with a high odds ratio for chronic kidney disease (OR = 2.70, 95% CI = 1.98-3.72) and low odds ratio for diabetes mellitus (DM) (OR = 0.61, 95% CI = 0.39-0.95). Comparison with 11 plasma biomarkers for age-related disease showed that plasma EC-SOD concentration correlated with adiponectin and estimated glomerular filtration rate with creatinine correction; therefore, we deduced that EC-SOD co-operates with adiponectin and possesses beneficial functions for DM in the Oldest Old.

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.

Superoxide dismutase (SOD) 3, a copper (Cu)-containing anti-oxidative enzyme, plays a key role in extracellular redox homeostasis. Cu chaperone antioxidant-1 (Atox-1) not only delivers Cu ions to SOD3 at the trans-Golgi network, it also functions as a transcription factor of SOD3; however, the role of Atox-1 in the regulation of SOD3 during the monocytic differentiation of THP-1 cells has not yet been elucidated. A treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the expression of the Cu transport protein ATP7A in THP-1 cells. On the other hand, the nuclear translocation of Atox-1 was detected in TPA-treated THP-1 cells, and was suppressed in the presence of the Cu chelator, bathocuproinedisulfonic acid. Furthermore, Atox-1 bound to the SOD3 promoter region in TPA-treated THP-1 cells. The overexpression of Atox-1 in THP-1 cells significantly enhanced TPA-elicited SOD3 expression, whereas its knockdown suppressed this induction. The present results demonstrate that Atox-1 functions as a key molecule in TPA-elicited SOD3 expression.

Superoxide dismutase (SOD) 3, one of the SOD isozymes, plays a pivotal role in extracellular redox homeostasis. The expression of SOD3 is regulated by epigenetics in human lung cancer A549 cells and human monocytic THP-1 cells; however, the molecular mechanisms governing SOD3 expression have not been elucidated in detail. Ten-eleven translocation (TET), a dioxygenase of 5-methylcytosine (5mC), plays a central role in DNA demethylation processes and induces target gene expression. In the present study, TET1 expression was abundant in U937 cells, but its expression was weakly expressed in A549 and THP-1 cells. These results are consistent with the expression pattern of SOD3 and its DNA methylation status in these cells. Moreover, above relationship was also observed in human breast cancer cells, human prostate cancer cells, and human skin fibroblasts. The overexpression of TET1-catalytic domain (TET1-CD) induced the expression of SOD3 in A549 cells, and this was accompanied by the direct binding of TET1-CD to the SOD3 promoter region. Furthermore, in TET1-CD-transfected A549 cells, the level of 5-hydroxymethylcytosine within that region was significantly increased, whereas the level of 5mC was decreased. The results of the present study demonstrate that TET1 might function as one of the key molecules in SOD3 expression through its 5mC hydroxylation in A549 cells.

Reactive oxygen species (ROS) produced by endothelial cells and macrophages play important roles in atherogenesis because they promote the formation of oxidized low-density lipoproteins (oxLDL). Extracellular-superoxide dismutase (EC-SOD) is mainly produced by vascular smooth muscle cells (VSMCs), is secreted into the extracellular space, and protects cells from the damaging effects of the superoxide anion. Thus, the expression of EC-SOD in VSMCs is crucial for protecting cells against atherogenesis; however, oxLDL-induced changes in the expression of EC-SOD in VSMCs have not yet been examined. We herein showed that oxLDL decreased EC-SOD mRNA and protein levels by binding to lectin-like oxidized LDL receptor-1 (LOX-1). Moreover, we demonstrated the significant role of mitogen-activated protein kinase (MEK)/extracellular-regulated protein kinase (ERK) signaling in oxLDL-elicited reductions in the expression of EC-SOD and proliferation of VSMCs. The results obtained with the FCS treatment indicate that oxLDL-elicited reductions in the expression of EC-SOD are related to the proliferation of VSMCs. We herein showed for the first time that luteolin, a natural product, restored oxLDL-induced decreases in the expression of EC-SOD and proliferation of VSMCs. Collectively, the results of the present study suggest that oxLDL accelerates the development of atherosclerosis by suppressing the expression of EC-SOD and also that luteolin has potential as a treatment for atherosclerosis. J. Cell. Biochem. 117: 2496-2505, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

Extracellular-superoxide dismutase (EC-SOD), one of the SOD isozymes, is negatively regulated under hypoxic conditions, and decreases in its expression may exacerbate vascular diseases. Moreover, epigenetics, such as DNA methylation and histone modifications, are known to play a critical role in the progression of cancer, type 2 diabetes, and atherosclerosis. We previously investigated the involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK) in decreases in EC-SOD expression in hypoxic COS7 cells; however, the role of epigenetics in this process currently remains unknown. In the present study, we demonstrated that the hypoxia mimetic cobalt chloride (CoCl 2 ) decreased histone acetylation levels, and a pretreatment with 4-phenyl butyric acid (PBA), an inhibitor of histone deacetylase, significantly suppressed CoCl 2 -elicited histone deacetylation and decreases in EC-SOD. We found that CoCl 2 -elicited decreases in EC-SOD were accompanied by reductions in histone H3 acetylation levels within its promoter region. Furthermore, luteolin, a well-known flavonoid, significantly suppressed the CoCl 2 -elicited accumulation of ROS, p38-MAPK activation, and histone deacetylation. Collectively, the results of the present study showed for the first time that CoCl 2 decreases the expression of EC-SOD through its deacetylation and luteolin may be one of the seed compounds that maintain redox homeostasis, even under hypoxic conditions.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall and plays an important role in normal redox homeostasis. We previously showed the significant reduction or induction of EC-SOD during human monocytic U937 or THP-1 cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively; however, its cell-specific expression and regulation have not been fully elucidated. It has been reported that epigenetic factors, such as DNA methylation and histone modification, are involved in several kinds of gene regulation. In this study, we investigated the involvement of epigenetic factors in EC-SOD expression and determined high levels of DNA methylation within promoter and coding regions of EC-SOD in THP-1 cells compared to those in U937 cells. Moreover, treatment with a DNA methyltransferase inhibitor, 5-azacytidine, significantly induced the expression of EC-SOD in THP-1 cells, indicating the importance of DNA methylation in the suppression of EC-SOD expression; however, the DNA methylation status did not change during THP-1 cell differentiation induced by TPA. On the other hand, we detected histone H3 and H4 acetylation during differentiation. Further, pretreatment with histone acetyltransferase inhibitors, CPTH2 or garcinol, significantly suppressed the TPA-inducible EC-SOD expression. We also determined the epigenetic suppression of EC-SOD in peripheral blood mononuclear cells. Treatment with granulocyte macrophage colony-stimulating factor (GM-CSF)/granulocyte-CSF induced that expression. Overall, these findings provide novel evidence that cell-specific and TPA-inducible EC-SOD expression are regulated by DNA methylation and histone H3 and H4 acetylation in human monocytic cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Endothelium-bound extracellular superoxide dismutase (eEC-SOD), a major antioxidative enzyme in the vasculature, is involved in anti-atherogenesis by inhibiting low-density lipoprotein (LDL) oxidation. The objective was to investigate whether the polyphenol-rich juar tea had beneficial effects on LDL oxidation and eEC-SOD levels in patients with metabolic syndrome (MetS). A total of 20 men with MetS participated in a randomized cross-over trial, comparing consumption of five cups/day of juar tea with that of a polyphenol-poor tea, barley tea, for 4 weeks. Although there was no change in LDL oxidizability after consumption of either tea, juar tea significantly increased eEC-SOD levels by 16% (p < 0.05), whereas barley tea significantly decreased levels by 15% (p < 0.05). It is noteworthy that the changes in eEC-SOD were positively associated with those in LDL oxidizability after tea consumption (r(2) = 0.11, p < 0.05). Tea polyphenols may provide anti-atherosclerotic effects by inhibiting LDL oxidation through EC-SOD bound to the endothelium.

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.

The aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by the investigation of two possibilities: first, change of EC-SOD expression in the retina; and secondly, leakage of EC-SOD through the endothelial monolayer by the treatment with endoplasmic reticulum (ER) stress inducers because ER stress is known to be involved in the vascular impairment in diabetic retinopathy. Intravitreous injection of tunicamycin in mice increased the permeability of tracer dye across retinal blood vessels while the retinal EC-SOD mRNA level was not changed. The leakage of EC-SOD through the retinal endothelial cell layer was elevated by the treatment with thapsigargin or tunicamycin. The expression of claudin-5 was significantly decreased by the treatment with the ER stress inducers. These phenomena were significantly suppressed by the pre-treatment of endothelial cells with a chemical chaperone 4-phenylbutyric acid. Our observations suggest that ER stress leads to the down-regulation of claudin-5 among tight junction proteins and may induce the elevation of endothelial permeability and leakage of EC-SOD into the vitreous body.

Endothelial dysfunction in essential hypertension is an independent predictor for future cardiovascular events. Although inhibition of the renin-angiotensin system (RAS) reportedly improves endothelial function through its effects on oxidative stress and inflammation, questions remain regarding the factors that are pivotal for improvement of endothelial function by RAS inhibition. We therefore performed a prospective, randomized crossover trial in which an angiotensin II type 1 receptor antagonist, olmesartan and calcium channel blocker, amlodipine, were compared in 31 essential hypertensive patients. Results showed that, although both treatments achieved comparable lowering of blood pressure (BP), olmesartan, but not amlodipine, significantly improved endothelial function as evaluated by flow-mediated vasodilation (FMD) in the brachial artery. Although no significant changes in diabetic and lipid parameters were observed with either drug, olmesartan slightly decreased estimated glomerular filtration rate, which, surprisingly, translated into decreased microalbuminuria. In a similar vein, olmesartan reduced serum C-reactive protein and increased urine antioxidant levels compared with baseline, and reduced urine 8-epi-prostaglandin F2α levels compared with both baseline and amlodipine. Finally, although overall changes in plasma extracellular superoxide dismutase (EC-SOD) levels were not modulated by either treatment, for olmesartan there was a positive correlation between changes in FMD and those in EC-SOD levels. In conclusion, olmesartan improved endothelial function in hypertensive patients independent of its BP-lowering effect, which was due, at least in part, to its antioxidative property. Therefore, olmesartan might provide a greater long-term benefit for hypertensive patients with impaired endothelial function than amlodipine.

It has been reported that tubular cells suffer an endoplasmic reticulum (ER) stress during the development of chronic kidney disease, which is a potent risk factor of cardiovascular disease. Moreover, under these conditions, reactive oxygen species are generated and induce cell injury. Extracellular-superoxide dismutase (EC-SOD) is a member of SODs and protects the cells from oxidative stress. Here, it is demonstrated that thapsigargin, an ER stress inducer, decreased EC-SOD expression, whereas the expression of Cu,Zn-SOD and Mn-SOD was not changed. On the other hand, another ER stress inducer, tunicamycin, did not affect the expression of EC-SOD. Further, it was shown that thapsigargin has the ability to activate extracellular-signal regulated kinase (ERK), but tunicamycin does not. Moreover, pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK, suppressed thapsigargin-triggered EC-SOD reduction, suggesting that MEK/ERK signalling should play an important role in the regulation of EC-SOD in COS7 cells under ER stress conditions.

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.

Cadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactive oxygen species (ROS) and induces cytotoxicity. Extracellular-superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the cells from damaging effects of ROS; however, the effect of Cd on the expression of EC-SOD in COS7 cells remains unclear. In this study, exposure to cadmium chloride (CdCl₂) enhanced intracellular ROS generation and induced COS7 cell death. Moreover, exposure to Cd decreased the expression of EC-SOD at mRNA and protein levels, but not of other SOD isozymes, copper-and zinc-containing SOD and manganese-containing SOD. The reduction of EC-SOD and cell viability was partially attenuated by pretreatment with an antioxidant, N-acetylcysteine. Further, we determined the involvement of p38-mitogen-activated protein kinase (p38-MAPK) in the reduction of EC-SOD. From these observations, p38-MAPK signaling cascades activated by ROS play a pivotal role in the reduction of EC-SOD, and it is concluded that the reduction of EC-SOD leads to a decrease in the resistance to oxidative stress of Cd-exposed COS7 cells.

The initial clinical stage of diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. The increased formation of reactive oxygen species (ROS) is thought to be a key event in the pathogenesis of DR. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that is distributed mainly in vascular cells and protects cells from ROS by scavenging superoxide anion. Treatment with cobalt chloride (CoCl(2)) decreased the expression of EC-SOD but not other SOD isozymes in pericytes accompanied with an increase of intracellular ROS production. Pre-treatment with N-acetylcysteine (NAC) significantly suppressed the ROS production and down-regulation of EC-SOD. We observed the activation of caspase-3 and DNA fragmentation as signs of apoptotic process by CoCl(2) treatment. In addition, these phenomena were significantly inhibited by pre-treatment with NAC. EC-SOD enhancer 4-phenyl butyric acid also suppressed the caspase-3 activation. It is known that the presence of a high level of EC-SOD throughout the vessel walls might have an important protective role against superoxide in the vascular system. The decrease in EC-SOD expression accompanied with elevation of ROS level in pericytes under hypoxia might induce and/or promote the ROS-triggered apoptosis of pericytes and the development of pathogenesis in DR.

Aim: The anti-oxidant enzyme copper/zinc superoxide dismutase (CuZnSOD) metabolizes superoxide anion (O(2)(-)) in vascular cells. However, the role of CuZnSOD in vascular injury remains poorly understood., Methods: Using CuZnSOD-deficient (CuZnSOD(-/-)) mice and wild-type (WT) mice, we investigated morphometric changes and the role of O(2)(-) in vascular remodeling after femoral artery injury induced by an external vascular cuff model., Results: Three days post-injury, inflammatory cell infiltration increased significantly. Moreover, the percent positive area of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in media were higher in CuZnSOD(-/-) mice than in WT mice (TNF-α: 34.8±8.4% versus 18.8±5.6%, p < 0.05, ICAM-1: 29.6±6.5% versus 11.0±2.8%, p < 0.05, VCAM-1: 23.5±7.5% versus 3.7±1.1%, p < 0.05). mRNA expression of iNOS was markedly increased in CuZnSOD(-/-) mice with cuff injury. Dihydroethidine staining revealed increased levels of vascular O(2)(-) in media from CuZnSOD(-/-) mice. Although neointimal formation remained unchanged, 14 days postinjury, we observed degeneration of the media, and the media/vessel wall ratio increased in CuZnSOD(-/-) mice (40.4±2.1% versus 26.8±1.4%, p < 0.05). Furthermore, SMemb/MHC-B-stained lesions increased markedly in CuZnSOD(-/-) mice., Conclusions: CuZnSOD-deficiency promoted inflammation, expressed adhesion molecules, and altered the structure of the media post-injury. Our results suggest that O(2)(-) participates importantly in the progression of early stage vascular inflammation, resulting in vascular remodeling in media but not neointimal formation, post-injury.

Diabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stress have been identified in the retina. Pericytes are known to be susceptible to oxidative stress and selective dropout of pericytes is one of the earliest pathological changes in DR. Extracellular-superoxide dismutase (EC-SOD) is a major antioxidative enzyme and protects vascular cells from the damaging effects of superoxide. Treatment with own conditioned medium significantly decreased EC-SOD expression in pericytes, while the expression of vascular endothelial growth factor and tumor necrosis factor-α were elevated. The addition of chemical chaperone 4-phenyl butyric acid significantly suppressed the effects of conditioned medium on EC-SOD and GRP78, a prominent ER-resident chaperone. Moreover, the cell viability of pericytes changed in a manner similar to that of EC-SOD expression. These results suggest that the expressions of EC-SOD should be regulated, at least partially, through ER stress. Continuous flow of culture media neutralized the ER-stress triggered decrease of EC-SOD expression. The stagnation of factors related to ER-stress around pericytes might reduce EC-SOD expression under pathophysiological conditions such as retinal edema, and this could induce and/or promote the intraretinal microvascular impairment and development of pathogenesis in DR.

It is well known that adipose tissue is not only a passive reservoir for energy storage but also produces and secretes a variety of bioactive molecules called adipocytokines, including adiponectin and tumor necrosis factor-alpha (TNF-alpha). Recently, it has been reported that adipose tissue can suffer a chronic hypoxic condition during hypertrophy of adipocytes, and this condition leads to the dysregulation of adipocytokines. Further, hypoxic adipocytes are in an increased oxidative stress. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from reactive oxygen species (ROS) by scavenging superoxide anion. Previous reports showed that plasma EC-SOD levels in type 2 diabetes patients were significantly and inversely related to the body mass index, homeostasis model assessment-insulin resistance index; however, the mechanisms of EC-SOD and adiponectin reductions during hypoxia remain poorly understood. Here, we demonstrate that cobalt chloride (CoCl(2)), a hypoxia mimetic, decreases EC-SOD and adiponectin in 3T3-L1 adipocytes by intracellular ROS-independent, but TNF-alpha and c-jun N-terminal kinase (JNK)-dependent mechanisms. From these results, it is possible that TNF-alpha is a key regulator of the reduction of EC-SOD and adiponectin in CoCl(2)-treated 3T3-L1 adipocytes, and we speculated that the reduction of EC-SOD and adiponectin would lead to and/or promote metabolic disorders.

Purpose: To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular superoxide dismutase (EC-SOD) in vitreous body and serum in patients with proliferative diabetic retinopathy (PDR), and investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. To investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model., Methods: EC-SOD and VEGF concentrations in vitreous and serum samples from PDR and macular hole (MH) were measured by ELISA. The effects of EC-SOD on VEGF-induced proliferation, migration, and tube formation were evaluated using human umbilical vein endothelial cells (HUVECs). Moreover, the effects of EC-SOD on VEGF-induced proliferation and migration were evaluated in HUVECs and primary normal human retinal microvascular endothelial cells., Results: Intravitreal concentrations of EC-SOD were significantly higher (p<0.01) in PDR (58.0+/-23.8 ng/ml, mean+/-SD) than in MH (29.3+/-6.6 ng/ml). Intravitreal concentrations of VEGF were dramatically higher (p<0.01) in PDR (798.2+/-882.7 pg/ml) than in MH (17.7+/-15.5 pg/ml). The serum concentrations of EC-SOD and VEGF did not differ between the two patient groups. The vitreous concentrations of VEGF correlated with those of EC-SOD in all patients (rs=0.61, p<0.001). In HUVECs, EC-SOD at 100 ng/ml significantly suppressed VEGF-induced proliferation and tube formation, but not VEGF-induced migration., Conclusions: EC-SOD was increased together with VEGF in the vitreous body from PDR patients, suggesting that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis.

Obesity is known to be the primary causal component in metabolic syndrome. Adipocytes in obese patients exhibit increased oxidative stress via the activation of reactive oxygen species (ROS)-producing systems and inactivation of antioxidant enzymes. Extracellular superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that protects cells from the damaging effects of ROS. An earlier report showed that plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to body mass index and homeostasis model assessment-insulin resistance index. Moreover, the administration of pioglitazone, an antidiabetic agent, significantly increased the plasma level of EC-SOD. In this report, the expression of EC-SOD was compared to other adipocytokines in mice 3T3-L1 pre-adipocytes. EC-SOD expression levels were increased after the induction of differentiation and then declined, which was similar to adiponectin and transcription factors such as peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha). On the other hand, the expression levels of pro-inflammatory adipocytokines, such as tumor necrosis factor-alpha (TNF-alpha) and monocyte chemo-attractant protein-1 (MCP-1), increased markedly in the development stage of cells. It was observed that the expression of EC-SOD in differentiated 3T3-L1 cells co-cultured with LPS-stimulated J774 macrophages was up-regulated, while the addition of TNF-alpha down-regulated EC-SOD and adiponectin expression in adipocytes. It is known that infiltrated and activated macrophages produce extracellular ROS at high levels in adipose tissue. It is possible that the expression of EC-SOD in adipocytes was stimulated to protect them from oxidative stress in the co-culture system.

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl(2))-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl(2).

Rationale: 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., Objectives: 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., Methods: MCT-injected rats were intratracheally administered vehicle (MCT group) or an adenovirus encoding beta-galactosidase (Adbetagal group) or human EC-SOD (AdEC-SOD group)., Measurements and Main Results: 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., Conclusions: 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.

Objective: Extracellular superoxide dismutase (EC-SOD) is the major extracellular scavenger of superoxides, and one of the main regulators of nitric oxide bioactivity in vessel walls. Here, we examined whether plasma EC-SOD level was associated with vasospastic angina (VSA), and if it was a risk factor for VSA., Methods and Results: We assigned 105 patients with normal or mildly stenotic coronary arteries into either a VSA (n=58) or chest pain syndrome (CPS) (n=47) groups. Plasma EC-SOD and other biochemical variables were measured, and major coronary risk factors were assessed. Results showed that apart from smoking status there were no significant differences in patient characteristics and biochemical variables between the two groups. In the VSA group, prevalence of smoking was significantly higher (53% versus 26%, p=0.0055), and plasma EC-SOD level was significantly lower (68.9+/-18.5 ng/ml versus 83.8+/-25.9 ng/ml; p=0.0009). Not only smoking (OR 2.742, 95% CI 1.032-7.287, p=0.0431) but also plasma EC-SOD (OR 0.971, 95% CI 0.949-0.993, p=0.0102) was an independent risk factor for VSA., Conclusions: In patients with VSA, plasma EC-SOD level was substantially reduced. Furthermore, plasma EC-SOD level followed by cigarette smoking was the most predictive risk factor for coronary spasms.

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in blood vessel walls, normal cartilage and synovial fluid and may be important for the antioxidant capability of these tissues. We have reported that EC-SOD gene transferred mice exhibited significant suppression of clinical symptoms of type II collagen induced arthritis [Iyama, et al., Arthritis Rheum., 44, 2160-2167 (2001)] and plasma EC-SOD levels in type 2 diabetic patients were significantly negatively related to indices of insulin resistance [Adachi, et al., J. Endocrinol., 181, 413-417 (2004)]. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathological conditions of the above diseases and is a major therapeutic target, based on clinical studies with anti-TNF-alpha monoclonal antibodies such as infliximab. In this report, we investigated the effect of TNF-alpha on the expression of EC-SOD in cultured cells and the cooperating effect of infliximab. In the in vitro assays examined, expression of EC-SOD, but not other SOD isozymes, in smooth muscle and fibroblast cells were suppressed by the addition of TNF-alpha. Simultaneous addition of infliximab dose-dependently and significantly prevented the suppressive effects of TNF-alpha. p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, prevented significantly the suppressive effect of TNF-alpha suggesting that p38 MAPK is an important signaling molecule downstream of TNF-alpha to inhibit the EC-SOD expression. From the results, it is speculated that the decline in TNF-alpha activity by the administration of infliximab results in the liberation of EC-SOD from the suppressed state of gene expression. This reveals a potential usefulness of infliximab on TNF-alpha related pathological conditions such as arthritis and insulin resistance.

Objectives: We studied whether the amount of heparin-released extracellular superoxide dismutase (EC-SOD), which is an antioxidative enzyme, is associated with coronary artery disease (CAD)., Methods and Results: EC-SOD was measured in plasma at basal and at post-heparin injection in 315 patients. Heparin-released EC-SOD was calculated as the difference between the two values. After exclusion of a mutant EC-SOD group (n = 27:8.6%), 288 patients were divided into three groups by angiographic findings; those with normal coronary (the normal group; n = 63), those with atherosclerosis without significant stenosis (the mild atherosclerosis group; n = 36), and those with significant stenosis (the atherosclerosis group; n = 189). Although the basal values were similar among the three groups, heparin-released EC-SOD levels were significantly lower in the atherosclerosis group (131.0 +/- 42.8 ng/ml, p = 0.0003) than in the normal group (156.9 +/- 66.2 ng/ml). Moreover, logistic analysis revealed that heparin-released EC-SOD independently contributed to CAD. The coronary score showed a significant correlation with heparin-released EC-SOD. As for factors affecting the level of heparin-released EC-SOD, the level of high-density lipoprotein cholesterol and age showed a positive correlation., Conclusions: The results suggest that heparin-released EC-SOD is significantly reduced in CAD and that the tissue-bound location of this enzyme might be important for antioxidative function.

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

The mechanisms of neointimal formation in cuff-injury models are still uncertain. To examine whether extracellular superoxide dismutase (EC-SOD) can reduce neointimal formation in a cuff-injury model, adenoviruses expressing EC-SOD (AxCAEC-SOD) or Escherichia coli beta-galactosidase (AxCALacZ) was injected between the cuff and the adventitia of rat femoral arteries. As a result, EC-SOD protein was effectively produced in the adventitia, as assessed by immunohistochemical staining. In comparison with cuff-treated control arteries and AxCALacZ-transfected arteries, neointimal formation was significantly reduced in AxCAEC-SOD-transfected arteries. Furthermore, proliferating smooth muscle cells in neointima and media were reduced by EC-SOD treatment. Similarly, augmented iNOS expression, apoptosis and collagen content in the vascular wall were also reduced by EC-SOD treatment. Reactive oxygen species (ROS) generation in tissue was reduced by EC-SOD expression, as assessed by dihydroethidium staining and coelenterazine chemiluminescence. These results suggest that ROS, especially superoxide anions at an adventitia, are responsible for neointimal formation in a cuff-injury model.

To study the protective function against oxygen radicals in the mesangial area, we assessed extracellular superoxide dismutase (EC-SOD) production in mesangial cells (MCs) in vitro. These cells have a major protective function against oxygen radicals in the extracellular space. In two different kinds of culture conditions: "growth medium" with fetal cow serum, and "differentiation medium" with reduced growth factor, and four extracellular matrixes; type I collagen, type IV collagen, laminin and fibronectin, were added to the MC culture. With the difference in the culture media, differentiation medium induced EC-SOD hyper-production associated with the both of the slowing down of cell proliferation and the suppression of IL-6 and IL-8 production. With difference in the extracellular matrix, the presence of type VI collagen and laminin promoted higher production of EC-SOD than fibronectin and type I collagen. Type IV collagen and laminin associated with the physiological condition of the glomeruli promoted EC-SOD production compared with the presence of type I collagen and fibronectin dominantly located in pathological condition. Suppression of EC-SOD production in growth medium along with MC proliferation and chemokine hyper-production compared with production in differentiation medium might mimic reduction of the protective capacity against oxygen radical toxity during mesangial proliferation in the glomerular nephritis. MC proliferation with type I collagen and fibronectin might enhance oxygen radical toxity in the glomeruli, and accelerate glomerular sclerosis through the suppression of EC-SOD production.

The purpose of this study was to investigate the relationship between exercise-induced oxidative stress and the menstrual cycle in healthy sedentary woman. Eighteen women with regular menstrual cycles participated in this research. The subjects monitored their basal body temperature (BBT) and carried out a urinary ovulation test (twice) for 2 months prior to the study to determine their menstrual cycle. The subjects performed bicycle ergometer exercise (for 30 min at 60% V(.)>O(2max)) in each phase (menses, follicular and luteal phases) of the menstrual cycle. Serum estradiol and progesterone concentrations were determined from blood that was collected at rest. Serum thiobarbituric acid reactive substances (TBARS), total superoxide dismutase (T-SOD) and extracellular superoxide dismutase (EC-SOD) were determined as markers of oxidative stress in blood samples collected at rest and after exercise. TBARS was significantly lower after exercise [2.4 (0.5) nmol/ml] in the follicular phase, and T-SOD was significantly lower after exercise [3.2 (1.2) U/ml] in the luteal phase. EC-SOD did not show a significant change after exercise during each phase of the menstrual cycle. Furthermore, there was a negative correlation between estradiol and DeltaT-SOD ( r=-0.46, P<0.05) and between estradiol and DeltaEC-SOD ( r=-0.55, P<0.05) during the menses. All data are presented as the mean value and its standard deviation. The results of this study suggest that when the estradiol level is high in a menstrual cycle, free radicals produced as a consequence of exercise may be easily eliminated by sedentary women with normal menstrual cycles.

Background: Homocysteine is involved in coronary atherosclerosis through oxidative stress, so the present study investigated the association between plasma concentrations of homocysteine and extracellular superoxide dismutase (EC-SOD) in coronary artery disease (CAD)., Methods and Results: The study group comprised 154 consecutive male patients with suspected CAD who had undergone angiography. Plasma concentrations of homocysteine and EC-SOD, which was determined before (basal) and after heparin therapy, were measured and the difference was designated as endothelium-bound EC-SOD. The EC-SOD ratio (endothelium-bound/basal EC-SOD) was also evaluated as an index of binding capacity. The plasma homocysteine concentration in the stenosis (+) group (n=97, 12.0+/-4.6 micromol/L) was significantly higher than that of the stenosis (-) group (n=57, 10.2+/-3.0 micromol/L, p=0.004). Plasma homocysteine correlated positively with the basal EC-SOD (r=0.377, p<0.001) and negatively with the EC-SOD ratio (r=-0.199, p=0.014). When the group was subdivided according to either homocysteine or the EC-SOD ratio, there were 2 groups with high homocysteine concentration and of these atherosclerosis was reduced in the group with a high EC-SOD ratio., Conclusions: In CAD patients, homocysteine is involved in the significant release of EC-SOD from the endothelium. Furthermore, the higher EC-SOD binding capacity, even at high concentrations of homocysteine, suggested that homocysteine-induced atherosclerosis was suppressed.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein located in blood vessel walls at high levels and may be important in the antioxidant capability of vascular walls. The aim of this study was to assess plasma levels of EC-SOD and to evaluate the relationship of the EC-SOD level with insulin resistance in type 2 diabetic patients. We determined plasma EC-SOD in 122 patients and found for the first time that the EC-SOD level was strongly and positively related to adiponectin (r=0.503, P < 0.001), and significantly and inversely related to fasting plasma glucose (FPG) (r=-0.209, P=0.022), body-mass index (BMI) (r=-0.187, P=0.040) and homeostasis model assessment-insulin resistance index (HOMA-R) (r=-0.190, P=0.039). Stepwise-multiple regression analysis also showed a significant influence of adiponectin (F=33.27) on the EC-SOD level. Administration of pioglitazone to 19 diabetic patients significantly increased the plasma levels of EC-SOD (69.9+/-19.3 ng/ml to 97.4+/-25.9 ng/ml; P < 0.0001) and adiponectin, while it decreased tumor necrosis factor-alpha (TNF-alpha). The present observations suggest that factors related to the pathogenesis of insulin resistance play an important role in the regulation of the plasma EC-SOD concentration. It is possible that the increase in the EC-SOD level by pioglitazone administration in diabetic patients is due to a decline of TNF-alpha, which is known to suppress EC-SOD expression.

Objective: We examined the vascular expression levels of extracellular superoxide dismutase (EC-SOD), a major antioxidant enzyme in the cardiovascular system, in patients with acute coronary syndromes., Methods and Results: Twenty-one consecutive patients with acute myocardial infarction (AMI), 14 patients with unstable angina, 11 patients with stable angina, and 20 control subjects were studied. The levels of vascular EC-SOD expression were assessed by the difference in plasma EC-SOD concentrations before and after intravenous heparan injection. In the patients with AMI, vascular EC-SOD expression (ng/mL) was significantly higher on day 1 after the onset of AMI (148+/-10) as compared with the control subjects (116+/-6, P<0.05). The vascular EC-SOD expression returned to the normal range on day 7 (104+/-8), and that level persisted thereafter. The vascular EC-SOD expression was also significantly higher in the patients with unstable angina (160+/-13) than in those with stable angina (122+/-10) or in the controls (116+/-6) (P<0.05 each). Moreover, in the patients with AMI, higher levels of vascular EC-SOD expression on day 1 were significantly associated with smaller myocardial infarct size (P<0.05)., Conclusions: This is the first clinical demonstration showing that vascular EC-SOD may be upregulated in acute coronary syndromes in humans in vivo. EC-SOD may play an important protective role against increased oxidative stress during acute ischemic coronary events.

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in the blood vessel walls and may be important for antioxidant capability of the vascular walls. Expression of EC-SOD is known to be regulated by numerous substances such as cytokines and vasoactive factors. Recently, we found that the plasma EC-SOD levels in type 2 diabetic patients were significantly and inversely related to indices of insulin resistance, whereas they were strongly and positively related to adiponectin. Administration of pioglitazone significantly increased the plasma level of EC-SOD and adiponectin. Transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs) are known to regulate genes associated with insulin resistance. The aim of this study was to assess the contribution of these transcription factors on the EC-SOD level. We found that a C/EBPbeta enhancer, prolactin, significantly induced the EC-SOD mRNA and protein levels in cultured fibroblast cell lines, but PPARgamma ligands, pioglitazone and other thiazolidinedione agents did not. Deletion analysis of the EC-SOD promoter-luciferase construct showed that an important element responsible for prolactin is located between -242 and -178 in the promoter region of the EC-SOD gene in which a known C/EBPbeta-binding site is located. Increasing the EC-SOD expression by treatment with ligands of transcription factors might be one approach to ameliorate the pathological conditions of insulin resistance.

Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 +/- 1.9 (sham treated) vs. 9.5 +/- 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.

Objective: The aim of this study was to determine the distribution of serum extracellular superoxide dismutase (EC-SOD) concentrations in patients with type 2 diabetes and to assess whether increased EC-SOD concentration is associated with the development of diabetic vascular complications., Research Design and Methods: Serum EC-SOD concentrations were determined in 222 patients with type 2 diabetes and 75 healthy control subjects by an enzyme-linked immunosorbent assay. All subjects had the EC-SOD domain genotyped., Results: The serum EC-SOD concentrations showed a distinct bimodal distribution in both patients with diabetes and control subjects. All subjects with the high-level phenotype carried the Arg213Gly mutation. The frequency of this variant was similar in the diabetes and control groups. Within the group of subjects with the common EC-SOD phenotype, the serum EC-SOD concentration (mean +/- SE) was significantly higher in patients with type 2 diabetes (99.3 +/- 1.3 ng/ml) compared with the control subjects (68.4 +/- 2.3 ng/ml, P < 0.01). Stepwise multiple regression analysis of the data from the diabetic common phenotype group showed a significant relationship between serum EC-SOD concentration and duration of diabetes (F = 5.31), carotid artery intimal-media thickness (F = 8.24), and severity of nephropathy (F = 16.05) and retinopathy (F = 4.43)., Conclusions: We observed a strong relationship between the serum concentration of EC-SOD and the severity of both micro- and macrovascular diabetic complications. These findings suggest that serum EC-SOD concentration levels may be a marker of vascular injury, possibly reflecting hyperglycemia-induced oxidative injury to the vascular endothelium and decreased binding of EC-SOD to the vascular wall.

Background: Hemodialysis (HD) is a method of chronic therapy for patients with renal failure. Diabetic nephropathy is the most common underlying disease among HD patients in Japan. A characteristic problem associated with this condition is endothelial cell damage. We have been using extracellular superoxide dismutase (EC-SOD) attached to the heparan sulfate on the endothelial cell surface as a marker of vascular damage., Methods: This study examined the pre- and post-HD levels of lipoprotein lipase (LPL). The increase in free fatty acid (FFA) is affected by a type of cytokine-like substances, which induces insulin resistance and causes abnormal lipid metabolism., Results: Pre-HD blood samples showed highly significant correlations between EC-SOD and LPL mass (r = 0.792) and between EC-SOD and FFA (r = 0.675) (p < 0.0001). EC-SOD, LPL mass, and FFA were remarkably high among the patients who had been placed on HD treatment for over 20 years., Conclusion: Because EC-SOD and LPL mass represent heparin-binding proteins, these results were considered to reflect severe vascular damage., (Copyright 2002 Elsevier Science B.V.)

Extracellular-superoxide dismutase (EC-SOD) [EC 1.15.1.1] is a secretory glycoprotein with an affinity for heparin-like proteoglycans. This enzyme locates in blood vessel walls at high levels and may be important for the antioxidant capability of vascular walls. Oxidative process plays an important role in atherogenesis. Lysophosphatidylcholine (lysoPC) is generated during oxidation of low-density lipoprotein (LDL) and is located within atherosclerotic plaques. Recently, lysoPC has been reported to induce transcription of a variety of cellular genes. In this study, we observed that lysoPC significantly increased the expression of EC-SOD mRNA and protein in human monocytic U937 cells, but not those of CuZn-SOD or Mn-SOD. Induced EC-SOD by lysoPC had a high affinity for heparin, and may bind to the endothelial cell surface. Very recently, it has been reported that exogenous addition of EC-SOD or overexpression of EC-SOD prevented endothelial cell-mediated oxidative modification of LDL. Therefore, it is speculated that EC-SOD is induced by lysoPC-stimulated monocytes as a feedback mechanism in vascular homeostasis.

Extracellular superoxide dismutase (EC-SOD) is synthesized in mesenchymally derived cells and prevents the oxygen radical-induced injury. We studied whether kidney mesangial cells (MCs) produce EC-SOD and how its production is associated with chemokine secretion. Under unstimulated condition, MCs produced EC-SOD, and its production was correlated positively with cyclic adenosine monophosphate (cAMP), but negatively with interleukin (IL)-6 or IL-8 production. By prednisolone or phorbol myristate acetate treatment, EC-SOD levels were correlated negatively with levels of IL-6 and IL-8. The presence of adenylate cyclase inhibitor 2',3'-dideoxyadenosine lost the prednisolone effect. The stimulation of EC-SOD production might be one of the important effects of prednisolone via cAMP pathway in MCs.

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in the arterial wall and may be important for antioxidation capability of the vascular wall and normal vascular function. EC-SOD is expressed in various cell types in the vascular wall such as fibroblasts, smooth muscle cells and macrophages, and the synthesis of EC-SOD by human fibroblasts is known to be highly responsive to various inflammatory cytokines, although there is no response to oxidative stress. Heparin is a highly sulfated glycosaminoglycan with many functions such as antithrombotic, antilipemic and antiatherosclerotic effects. Another less well-known function of heparin is regulation of protein synthesis. In this study, we measured the induction of EC-SOD after treatment with heparin to understand the role of heparin in the antiatherosclerotic response of fibroblasts. Heparin induced EC-SOD expression at both the mRNA and protein levels. Heparin showed the greatest stimulatory effect and heparan sulfate showed moderate effects. The effect of chondroitin sulfate A was not clear. In contrast, desulfated heparin and chondroitin sulfate C did not increase EC-SOD expression. The stimulatory effect seemed to increase roughly with the degree of glycosaminoglycan sulfation. The enhanced expression of EC-SOD by heparin must contribute to the antiatherosclerotic effect of heparin.

Extracellular-superoxide dismutase (EC-SOD) is bound to the vascular endothelial cell surface with an affinity for heparan sulfate proteoglycan. The binding of EC-SOD to the human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell surface proteoglycans was significantly decreased by the incubation with S-nitroso-N-acetyl-DL-penicillamine (SNAP) and +/- -N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4), potent nitric oxide (NO) donors. NO derived from lipopolysaccharide-stimulated J774 A-1 cells also decreased the binding of EC-SOD to HUVEC, and this decrease was blocked by N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor. SNAP and NOR4 also decreased the binding of EC-SOD to immobilized heparin. Furthermore, the decomposed derivatives of NO donors and sodium nitrite decreased the binding of EC-SOD. These observations suggest that excess NO produced in the inflammatory conditions decreases the binding of EC-SOD to the vascular endothelial cell surface, which results in a loss of the ability to protect the endothelial cell surface from oxidative stress.

Unlabelled: OBJECTIVE; Superoxide dismutase (SOD) is a potent antiinflammatory enzyme that has received growing attention for its therapeutic potential. This study was undertaken to examine the efficacy of extracellular SOD (EC-SOD) gene therapy in murine collagen-induced arthritis., Methods: Embryonic DBA/1 mouse fibroblasts were infected with a recombinant retrovirus expressing human EC-SOD. DBA/1 mice that had been treated with type II collagen were administered subcutaneous injections of 2 x 10(7) EC-SOD-expressing fibroblasts on day 29, when symptoms of arthritis were already present. The severity of arthritis in individual mice was evaluated in a double-blind manner; each paw was assigned a separate clinical score, and hind paw thickness was measured with a caliper. Mice were killed on day 50 for histologic examination of the joints., Results: High serum concentrations of EC-SOD were maintained for at least 7 days. Mice treated with the transgene exhibited significant suppression of clinical symptoms such as disabling joint swelling, deformity, and hind paw thickness, compared with the untreated group (mean +/- SD maximum clinical score in the untreated and the transgene-treated groups 2.71 +/- 1.08 and 1.35 +/- 1.22, respectively; P < 0.01, and hind paw thickness 3.04 +/- 0.18 mm and 2.56 +/- 0.12 mm, respectively; P < 0.05). Histologic abnormalities, including destruction of cartilage and bone, infiltration of mononuclear cells, and proliferation of synovial cells, were also markedly improved in the EC-SOD-treated mice compared with the control group (histopathologic score 7.50 +/- 1.13 and 4.13 +/- 1.88 in the untreated and transgene-treated groups, respectively; P < 0.05)., Conclusion: These results indicate that EC-SOD gene transfer may be an effective form of therapy for rheumatoid arthritis.

Reactive oxygen species have been proposed to play important roles in atherosclerosis. To investigate the protective role of extracellular superoxide dismutase (EC-SOD), its inhibition of endothelial-cell-mediated LDL oxidation was examined. We constructed the recombinant adenovirus AxCAEC-SOD expressing human EC-SOD by CAG promoter. Infection of endothelial cells with AxCAEC-SOD resulted in EC-SOD protein secretion in a dose-dependent manner and a decrease of endothelial-cell-derived superoxide production. Moreover, it was proven to coexist with heparan sulfate by immunohistochemical staining. Endothelial-cell-mediated LDL oxidation enhanced by ferric-sodium EDTA was inhibited by 47% in TBARS formation by AxCAEC-SOD infection. In agarose gel electrophoresis, AxCAEC-SOD decreased the negative charge of oxidized LDL by 50% and suppressed fragmentation of apolipoprotein B. These results suggested that human EC-SOD localized in the extracellular space and reduced endothelial-cell-mediated LDL oxidation. In subendothelial space, EC-SOD bound on heparan sulfate might suppress LDL oxidation through reduction of superoxide anion., (Copyright 2001 Academic Press.)

Extracellular-superoxide dismutase (EC-SOD) is one of the three SOD isozymes in humans and has affinity for heparin-like sulfated glycosaminoglycans in the extracellular matrix. The C-terminal portion of EC-SOD is responsible for the heparin-affinity of this enzyme, but this portion should be a target of proteinases. Recently, Oury et al. reported that EC-SOD is localized within the cytoplasm of neuronal cells, while this enzyme is found in the extracellular matrix in most other tissues. It has been speculated that the heparin-binding domain of EC-SOD in the brain is sensitive to proteolysis, which may result in a loss of extracellular immunoreactivity. This study was performed to investigate the heparin-affinity of EC-SOD in the human brain. We found that human EC-SOD in brain tissue has high heparin-affinity similar to that in the umbilical cord. Moreover, heparin-affinity of EC-SOD in brain homogenate was not decreased by incubation at 37 degrees C for 72 h. The proteolytic activity in brain homogenate might be less than that in umbilical cord homogenates. The intracellular localization of EC-SOD could contribute to its slow clearance from the brain and maintenance of its physiological functions in the brain.

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 microM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.

A modest homocysteine elevation is associated with an increased cardiovascular risk. Marked circulating homocysteine elevations occur in homocystinuria due to cystathionine beta-synthase (CbetaS) deficiency, a disorder associated with a greatly enhanced cardiovascular risk. Lowering homocysteine levels reduces this risk significantly. Because homocysteine-induced oxidative damage may contribute to vascular changes and extracellular superoxide dismutase (EC-SOD) is an important antioxidant in vascular tissue, we assessed EC-SOD and homocysteine in patients with homocystinuria. We measured circulating EC-SOD, total homocysteine (free plus bound), and methionine levels during the treatment of 21 patients with homocystinuria, 18 due to CbetaS deficiency, aged 8 to 59 years, and 3 with remethylating defects. We measured total homocysteine by immunoassay, EC-SOD by ELISA, and methionine by amino acid analysis and assessed interindividual and intraindividual relationships. There was a significant, positive relationship between EC-SOD and total homocysteine. For the interindividual assessment, levels were highly correlated, r=0.746, N=21, P<0.0001. This relationship was maintained after taking into account intraindividual patient variation (r=0.607, N=62, P<0.0001). In 2 newly diagnosed CbetaS-deficient patients, treatment that lowered the markedly elevated pretreatment homocysteine level (from 337 to 72 and from 298 to 50 micromol/L) reduced the associated elevated EC-SOD in each by 50%. EC-SOD and methionine levels were unrelated (r=0.148, n=39, P=0.368). The positive relationship between circulating EC-SOD and homocysteine could represent a protective antioxidant response to homocysteine-induced oxidative damage and contribute to reducing cardiovascular risk in homocystinuric patients. EC-SOD levels may be relevant to the pathogenesis of vascular disease in other patient groups.