Heparin-induced release of extracellular superoxide dismutase to human blood plasma.
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ABSTRACT:
SUBMITTER: Karlsson K
PROVIDER: S-EPMC1147663 | biostudies-other | 1987 Feb
REPOSITORIES: biostudies-other
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Project description:The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.
| S-EPMC1134540 | biostudies-other
Project description:An important issue in critical care medicine is the identification of ways to protect the lungs from oxygen toxicity and reduce systemic oxidative stress in conditions requiring mechanical ventilation and high levels of oxygen. One way to prevent oxygen toxicity is to augment antioxidant enzyme activity in the respiratory system. The current study investigated the ability of aerosolized extracellular superoxide dismutase (EC-SOD) to protect the lungs from hyperoxic injury. Recombinant human EC-SOD (rhEC-SOD) was produced from a synthetic cassette constructed in the methylotrophic yeast Pichia pastoris. Female CD-1 mice were exposed in hyperoxia (FiO2>95%) to induce lung injury. The therapeutic effects of EC-SOD and copper-zinc SOD (CuZn-SOD) via an aerosol delivery system for lung injury and systemic oxidative stress at 24, 48, 72 and 96 h of hyperoxia were measured by bronchoalveolar lavage, wet/dry ratio, lung histology, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) in lung and liver tissues. After exposure to hyperoxia, the wet/dry weight ratio remained stable before day 2 but increased significantly after day 3. The levels of oxidative biomarker 8-oxo-dG in the lung and liver were significantly decreased on day 2 (P<0.01) but the marker in the liver increased abruptly after day 3 of hyperoxia when the mortality increased. Treatment with aerosolized rhEC-SOD increased the survival rate at day 3 under hyperoxia to 95.8%, which was significantly higher than that of the control group (57.1%), albumin treated group (33.3%), and CuZn-SOD treated group (75%). The protective effects of EC-SOD against hyperoxia were further confirmed by reduced lung edema and systemic oxidative stress. Aerosolized EC-SOD protected mice against oxygen toxicity and reduced mortality in a hyperoxic model. The results encourage the use of an aerosol therapy with EC-SOD in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure, including acute respiratory distress syndrome (ARDS).
| S-EPMC3202580 | biostudies-literature
Project description:The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.
| S-EPMC1132320 | biostudies-other
Project description:Disruption of the oxidant/antioxidant balance in the lung is thought to be a key step in the development of many airway pathologies. Hence, antioxidant enzymes play key roles in controlling or preventing pulmonary diseases related to oxidative stress. The superoxide dismutases (SOD) are a family of enzymes that play a pivotal role protecting tissues from damage by oxidant stress by scavenging superoxide anion, which prevents the formation of other more potent oxidants such as peroxynitrite and hydroxyl radical. Extracellular SOD (EC-SOD) is found predominantly in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. EC-SOD has been shown to be protective in several models of interstitial lung disease, including pulmonary fibrosis. In addition, alterations in EC-SOD expression are also present in human idiopathic pulmonary fibrosis (IPF). This review discusses EC-SOD regulation in response to pulmonary fibrosis in animals and humans and reviews possible mechanisms by which EC-SOD may protect against fibrosis.
| S-EPMC2290736 | biostudies-literature
Project description:PurposeSuperoxide is produced by activated neutrophils during the inflammatory response to stimuli such as endotoxin, can directly or indirectly injure host cells, and has been implicated in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We wished to determine the potential for pulmonary overexpression of the extracellular isoform of superoxide dismutase (EC-SOD) to reduce the severity of endotoxin-induced lung injury.MethodsAnimals were randomly allocated to undergo intratracheal instillation of (1) surfactant alone (vehicle); (2) adeno-associated virus (AAV) vectors containing a null transgene (AAV-null); and (3) adeno-associated virus vectors containing the EC-SOD transgene (AAV-EC-SOD) and endotoxin was subsequently administered intratracheally. Two additional groups were randomized to receive (1) vehicle or (2) AAV-EC-SOD, and to undergo sham (vehicle) injury. The severity of the lung injury was assessed in all animals 24 h later.ResultsEndotoxin produced a severe lung injury compared to sham injury. The AAV vector encoding EC-SOD increased lung EC-SOD concentrations, and enhanced the antioxidant capacity of the lung. EC-SOD overexpression decreased the severity of endotoxin-induced ALI, reducing the decrement in systemic oxygenation and lung compliance, decreasing lung permeability and decreasing histologic injury. EC-SOD attenuated pulmonary inflammation, decreased bronchoalveolar lavage neutrophil counts, and reduced interleukin-6 and CINC-1 concentrations. The AAV vector itself did not contribute to inflammation or to lung injury.ConclusionsPulmonary overexpression of EC-SOD protects the lung against endotoxin-induced ALI.
| S-EPMC7095197 | biostudies-literature
Project description:In the present study, we investigate the effect of homocysteine (Hcy) on extracellular-superoxide dismutase (EC-SOD) DNA methylation in the aorta of mice, and explore the underlying mechanism in macrophages, trying to identify the key targets of Hcy-induced EC-SOD methylation changes. ApoE -/- mice are fed different diets for 15 weeks, EC-SOD and DNA methyltransferase 1 (DNMT1) expression levels are detected by RT-PCR and western blot analysis. EC-SOD methylation levels are assessed by ntMS-PCR. After EC-SOD overexpression or knockdown in macrophages, following the transfection of macrophages with pEGFP-N1-DNMT1, the methylation levels of EC-SOD are detected. Our data show that the concentrations of Hcy and the area of atherogenic lesions are significantly increased in ApoE -/- mice fed with a high-methionine diet, and have a positive correlation with the levels of superoxide anions, which indicates that Hcy-activated superoxide anions enhance the development of atherogenic lesions. EC-SOD expression is suppressed by Hcy, and the content of superoxide anion is increased when EC-SOD is silenced by RNAi in macrophages, suggesting that EC-SOD plays a major part in oxidative stress induced by Hcy. Furthermore, the promoter activity of EC-SOD is increased following transfection with the -1/-1100 fragment, and EC-SOD methylation level is significantly suppressed by Hcy, and more significantly decreased upon DNMT1 overexpression. In conclusion, Hcy may alter the DNA methylation status and DNMT1 acts as the essential enzyme in the methyl transfer process to disturb the status of EC-SOD DNA methylation, leading to decreased expression of EC-SOD and increased oxidative stress and atherosclerosis.
| S-EPMC9827811 | biostudies-literature
Project description:The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.
| S-EPMC1151575 | biostudies-other
Project description:A further characterization of human extracellular superoxide dismutase is reported. The study was especially aimed at the interaction with substances known to interfere with CuZn superoxide dismutase and other superoxide dismutases. Extracellular superoxide dismutase is efficiently inhibited by cyanide and is about 3 times more sensitive than is human CuZn superoxide dismutase. The sensitivity to azide is much lower, but still about 3 times higher than that of human CuZn superoxide dismutase. Extracellular superoxide dismutase is about as rapidly inactivated by hydrogen peroxide as is CuZn superoxide dismutase. The sensitivity to diethyldithiocarbamate is very high and more than an order of magnitude larger than that of CuZn superoxide dismutase. Sodium dodecyl sulphate, under conditions suggested as being suitable for distinguishing between the insensitive CuZn superoxide dismutase and the sensitive Mn superoxide dismutase, efficiently inactivated extracellular superoxide dismutase. No antigenic similarities between extracellular superoxide dismutase and CuZn superoxide dismutase could be demonstrated. Anti-(extracellular superoxide dismutase) did not bind CuZn superoxide dismutase, and anti-(CuZn superoxide dismutase) did not bind extracellular superoxide dismutase.
| S-EPMC1153619 | biostudies-other
Project description:Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.
| S-EPMC3095923 | biostudies-literature
Project description:Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)-induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload.
| S-EPMC2429959 | biostudies-literature