Enzyme Extracellular Superoxide Dismutase Research Articles

Interleukin-1β triggers muscle-derived extracellular superoxide dismutase expression and protects muscles from doxorubicin-induced atrophy.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1β (IL-1β) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1β treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1β (IL-1β) expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

In vitro and in vivo Antiallergic Effects of Taurine on Allergic Rhinitis

Background: The current treatment for allergic rhinitis (AR) is inadequate. Objective: The present study aimed to investigate the therapeutic effect of taurine on AR and to identify the underlying molecular mechanisms. Methods: The serum level of the antioxidant enzyme extracellular superoxide dismutase (SOD3) was determined in AR patients and in healthy controls. The antiallergic inflammatory effects of taurine were evaluated in a dinitrophenyl-human serum albumin (DNP-HSA)-stimulated human mast cell line (HMC-1) and in an ovalbumin (OVA)-induced AR mouse model. Results: Clinically, a reduction in serum level of SOD3 was observed in AR patients. Taurine treatment led to dose-dependent increases in SOD3 at both protein and mRNA levels in HMC-1 cells. SOD3 production was regulated by peroxisome proliferator-activated receptor-γ (PPAR-γ) in response to taurine. SOD3 overexpression inhibited the release of proinflammatory cytokines including tumor necrosis factor-α (, interleukin (IL)-4, and IL-6. Its overexpression also ameliorated the loss of interferon-γ. SOD3 and PPAR-γ influenced inflammatory cytokine production via regulation of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). An OVA-induced AR animal model study showed that taurine was efficacious in alleviating allergic inflammatory reactions by relieving behavior symptoms of AR mice and reducing eosinophilic and mast cell infiltration into the nasal cavity. In addition, taurine treatment increased the production of SOD3 and PPAR-γ, which, in turn, suppressed expression of proinflammatory cytokines through phosphorylation of ERK1/2. Conclusion: Taurine could potentially serve as a therapeutic treatment for allergic disorders.

Extracellular Superoxide Dismutase Regulates Early Vascular Hyaluronan Remodeling in Hypoxic Pulmonary Hypertension

Chronic hypoxia leads to pathologic remodeling of the pulmonary vasculature and pulmonary hypertension (PH). The antioxidant enzyme extracellular superoxide dismutase (SOD3) protects against hypoxia-induced PH. Hyaluronan (HA), a ubiquitous glycosaminoglycan of the lung extracellular matrix, is rapidly recycled at sites of vessel injury and repair. We investigated the hypothesis that SOD3 preserves HA homeostasis by inhibiting oxidative and enzymatic hyaluronidase-mediated HA breakdown. In SOD3-deficient mice, hypoxia increased lung hyaluronidase expression and activity, hyaluronan fragmentation, and effacement of HA from the vessel wall of small pulmonary arteries. Hyaluronan fragmentation corresponded to hypoxic induction of the cell surface hyaluronidase-2 (Hyal2), which was localized in the vascular media. Human pulmonary artery smooth muscle cells (HPASMCs) demonstrated hypoxic induction of Hyal2 and SOD-suppressible hyaluronidase activity, congruent to our observations in vivo. Fragmentation of homeostatic high molecular weight HA promoted HPASMC proliferation in vitro, whereas pharmacologic inhibition of hyaluronidase activity prevented hypoxia- and oxidant-induced proliferation. Hypoxia initiates SOD3-dependent alterations in the structure and regulation of hyaluronan in the pulmonary vascular extracellular matrix. These changes occurred soon after hypoxia exposure, prior to appearance of PH, and may contribute to the early pathogenesis of this disease.

TLR4 is a critical regulator of angiotensin II-induced vascular remodeling: the roles of extracellular SOD and NADPH oxidase.

Toll-like receptor 4 (TLR4) and angiotensin II (AngII) induce vascular remodeling through the production of reactive oxygen species (ROS). AngII has also been shown to increase antioxidant enzyme extracellular superoxide dismutase (ecSOD). However, the roles of TLR4 in Ang II-induced ROS production, vascular remodeling and hypertension remain unknown. Mice lacking TLR4 function showed significant inhibition of vascular remodeling in response to chronic AngII infusion, with no impact on blood pressure. The increases in ROS level and NADPH oxidase activity in response to AngII infusion were markedly blunted in TLR4-deficient mice. Similar effects were observed in wild-type (WT) mice treated with a sub-depressor dose of the AT1 receptor antagonist irbesartan, which had no effects on TLR4-deficient mice. Intriguingly, the AngII infusion-induced increases in ecSOD activity and expression were rather enhanced in TLR4-deficient mice compared with WT mice, whereas the expression of the proinflammatory chemokine MCP-1 was decreased. Importantly, AngII-induced vascular remodeling was positively correlated with NADPH oxidase activity, ROS levels and MCP-1 expression levels. Notably, chronic norepinephrine infusion, which elevates blood pressure without increasing ROS production, did not induce significant vascular remodeling in WT mice. Taken together, these findings suggest that ROS elevation is required for accelerating vascular remodeling but not for hypertensive effects in this model. We demonstrated that TLR4 plays a pivotal role in regulating AngII-induced vascular ROS levels by inhibiting the expression and activity of the antioxidant enzyme ecSOD, as well as by activating NADPH oxidase, which enhances inflammation to facilitate the progression of vascular remodeling.

Pioglitazone prevents the endothelial dysfunction induced by ischemia and reperfusion in healthy subjects.

No study has investigated whether pioglitazone (an agonist of peroxisome proliferator-activated receptor gamma) protects against ischemia and reperfusion (IR)-induced endothelial dysfunction in humans. In the first crossover study, 20 volunteers were randomized to 1 week of pioglitazone (30 mg/d, postoperatively) or control (no treatment). In the second single-arm study, 15 volunteers received pioglitazone and the cyclooxygenase-2 inhibitor meloxicam for 1 week. On day 7, endothelium-dependent flow-mediated dilation (FMD) of the distal brachial artery was measured before and after IR (15 minutes of ischemia followed by 15 minutes of reperfusion in the proximal upper arm). Pre-IR brachial-artery diameter and FMD were similar across the 2 sessions (control, pioglitazone) in protocol 1 and between the 2 protocols. IR significantly blunted FMD after no treatment (pre-IR FMD: 10.2% ± 2.6%; post-IR FMD: 3.5% ± 1.9%, P < 0.01) but not after pioglitazone administration (pre-IR FMD: 9.7% ± 2.5%; post-IR FMD: 8.8% ± 2.9%, P = 0.11). This protective effect was accompanied by an increase in serum levels of the antioxidant enzyme extracellular superoxide dismutase and was not affected by concomitant administration of the cyclooxygenase-2 inhibitor meloxicam (P = 0.10). In humans, pioglitazone provides potent protection against IR-induced endothelial dysfunction.

A common polymorphism in extracellular superoxide dismutase affects cardiopulmonary disease risk by altering protein distribution.

The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single-nucleotide polymorphism in EC-SOD (rs1799895) leads to an arginine to glycine amino acid substitution at position 213 (R213G) in the heparin-binding domain. In recent human genetic association studies, this single-nucleotide polymorphism attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease. Capitalizing on the complete sequence homology between human and mouse in the heparin-binding domain, we created an analogous R213G single-nucleotide polymorphism knockin mouse. The R213G single-nucleotide polymorphism did not change enzyme activity, but shifted the distribution of EC-SOD from lung and vascular tissue to extracellular fluid (eg, bronchoalveolar lavage fluid and plasma). This shift reduces susceptibility to lung disease (lipopolysaccharide-induced lung injury) and increases susceptibility to cardiopulmonary disease (chronic hypoxic pulmonary hypertension). We conclude that EC-SOD provides optimal protection when localized to the compartment subjected to extracellular oxidative stress: thus, the redistribution of EC-SOD from the lung and pulmonary circulation to the extracellular fluids is beneficial in alveolar lung disease but detrimental in pulmonary vascular disease. These findings account for the discrepant risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.

Extracellular superoxide dismutase is important for hippocampal neurogenesis and preservation of cognitive functions after irradiation

Cranial irradiation is widely used in cancer therapy, but it often causes cognitive defects in cancer survivors. Oxidative stress is considered a major cause of tissue injury from irradiation. However, in an earlier study mice deficient in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD KO) showed reduced sensitivity to radiation-induced defects in hippocampal functions. To further dissect the role of EC-SOD in neurogenesis and in response to irradiation, we generated a bigenic EC-SOD mouse model (OE mice) that expressed high levels of EC-SOD in mature neurons in an otherwise EC-SOD-deficient environment. EC-SOD deficiency was associated with reduced progenitor cell proliferation in the subgranular zone of dentate gyrus in KO and OE mice. However, high levels of EC-SOD in the granule cell layer supported normal maturation of newborn neurons in OE mice. Following irradiation, wild-type mice showed reduced hippocampal neurogenesis, reduced dendritic spine densities, and defects in cognitive functions. OE and KO mice, on the other hand, were largely unaffected, and the mice performed normally in neurocognitive tests. Although the resulting hippocampal-related functions were similar in OE and KO mice following cranial irradiation, molecular analyses suggested that they may be governed by different mechanisms: whereas neurotrophic factors may influence radiation responses in OE mice, dendritic maintenance may be important in the KO environment. Taken together, our data suggest that EC-SOD plays an important role in all stages of hippocampal neurogenesis and its associated cognitive functions, and that high-level EC-SOD may provide protection against irradiation-related defects in hippocampal functions.

Highlights of This Issue

The role of G-protein coupled–receptors (GPCR) in cancer and how to best target their function remains incompletely understood. Using The Cancer Genome Atlas, primary tumor specimens, and in vitro studies, Woerner and coworkers found that expression of G-protein coupled–receptor kinases (GRK), critical regulators of GPCR function, is altered in glioblastoma in a molecular subtype–specific fashion. Decreased GRK3 expression in classical glioblastoma was associated with a growth advantage, which was blocked by reconstitution of GRK3 expression. These data indicate that GPCRs function to regulate glioblastoma growth and suggest that modulation of GPCR phosphorylation may be an important therapeutic strategy.The antioxidant enzyme extracellular superoxide dismutase EcSOD is downregulated in a large proportion of human lung cancers. To explore its role in cancer, Teoh-Fitzgerald and colleagues examined EcSOD gene expression, as well as the biological effects of its reexpression in EcSOD-negative tumor cells. Epigenetic silencing by aberrant cytosine methylation of the promoter was the most frequent cause of expression loss in human lung adenocarcinoma compared with normal lung. Reexpression of EcSOD inhibited the in vitro invasiveness of human lung adenocarcinoma cells. This study identifies EcSOD as an epigenetically regulated lung cancer target and a modifier of the malignant phenotype.TRAIL induces apoptosis in basal/triplenegative breast cancer (TNBC) cell lines, whereas other subtypes are relatively resistant. Inhibition of WEE1, a cell-cycle checkpoint regulator, causes increased cell death in cancer cell lines. Here, Garimella and colleagues show that pretreatment with WEE1 inhibitor increased the toxicity of TRAIL in TNBC cell lines. This effect is attributed to increased surface expression of death receptors resulting in increased caspase activation initiated by caspase-8. These data suggest that the combination of WEE1 inhibitor and TRAIL could provide a novel approach to targeted therapy for TNBC.Although largely unknown, the abnormalities in DNA repair that underlie the genomic instability in sporadic cancers are potential therapeutic targets. Tobin and colleagues have characterized a novel defect in DNA double-strand break repair in hormone therapy–resistant breast cancer that can be identified in breast cancer cell lines and tumor biopsies and can be selectively targeted by a combination of PARP and DNA ligase inhibitors. These preclinical studies provide the rationale for the development of effective therapeutic strategies for hormone therapy–resistant breast cancer that may be applicable to other cancers, including leukemias, with similar DNA repair abnormalities.

Leukocyte-derived extracellular superoxide dismutase does not contribute to airspace EC-SOD after interstitial pulmonary injury

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is known to limit inflammation and fibrosis following numerous pulmonary insults. Previous studies have reported a loss of full-length EC-SOD from the pulmonary parenchyma with accumulation of proteolyzed EC-SOD in the airspace after an interstitial lung injury. However, following airspace only inflammation, EC-SOD accumulates in the airspace without a loss from the interstitium, suggesting this antioxidant may be released from an extrapulmonary source. Because leukocytes are known to express EC-SOD and are prevalent in the bronchoalveolar lavage fluid (BALF) after injury, it was hypothesized that these cells may transport and release EC-SOD into airspaces. To test this hypothesis, C57BL/6 wild-type and EC-SOD knockout mice were irradiated and transplanted with bone marrow from either wild-type mice or EC-SOD knockout mice. Bone marrow chimeric mice were then intratracheally treated with asbestos and killed 3 and 7 days later. At both 3 and 7 days following asbestos injury, mice without pulmonary EC-SOD expression but with EC-SOD in infiltrating and resident leukocytes did not have detectable levels of EC-SOD in the airspaces. In addition, leukocyte-derived EC-SOD did not significantly lessen inflammation or early stage fibrosis that resulted from asbestos injury in the lungs. Although it is not influential in the asbestos-induced interstitial lung injury model, EC-SOD is still known to be present in leukocytes and may play an influential role in attenuating pneumonias and other inflammatory diseases.

Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.

The effects of aging on pulmonary oxidative damage, protein nitration, and extracellular superoxide dismutase down-regulation during systemic inflammation

Systemic inflammatory response syndrome (SIRS), a serious clinical condition characterized by whole-body inflammation, is particularly threatening for elderly patients, who suffer much higher mortality rates than the young. A major pathological consequence of SIRS is acute lung injury caused by neutrophil-mediated oxidative damage. Previously, we reported an increase in protein tyrosine nitration (a marker of oxidative/nitrosative damage) and a decrease in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in the lungs of young mice during endotoxemia-induced SIRS. Here we demonstrate that during endotoxemia, down-regulation of EC-SOD is significantly more profound and prolonged, whereas up-regulation of iNOS is augmented, in aged compared to young mice. Aged mice also showed 2.5-fold higher protein nitration levels, compared to young mice, with particularly strong nitration in the pulmonary vascular endothelium during SIRS. Additionally, by two-dimensional gel electrophoresis, Western blotting, and mass spectrometry, we identified proteins that show increased tyrosine nitration in age- and SIRS-dependent manners; these proteins (profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin) include components of the actin cytoskeleton responsible for maintaining pulmonary vascular permeability. Reduced EC-SOD in combination with increased oxidative/nitrosative damage and altered cytoskeletal protein function due to tyrosine nitration may contribute to augmented lung injury in the aged with SIRS.

Endogenous enzymes (NOX and ECSOD) regulate smoke-induced oxidative stress

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and the incidence is increasing as the population ages. Cigarette smoking is the primary risk factor; however, only a minority of smokers develop the disease. Inhalation of cigarette smoke introduces an abundance of free radicals into the lungs, causing oxidative stress and inflammation. We hypothesized that after the initial burst of oxidative stress associated with cigarette smoke exposure, a sustained source of endogenous free radical production is modulated by the antioxidant enzyme extracellular superoxide dismutase (ECSOD) and the superoxide-generating complex NADPH oxidase (NOX). Primary mouse macrophages exposed to cigarette smoke extract exhibited increased oxidative stress as indicated by fluorogenic dyes and isoprostane concentration, which was suppressed in the presence of both a superoxide dismutase mimetic and a NOX inhibitor. Similarly, primary macrophages isolated from ECSOD-overexpressing mice or NOX-deficient mice showed reduced oxidative stress in response to cigarette smoke treatment. In addition, both reduced glutathione and cytokines (MIP2 and IFNγ) were increased in bronchoalveolar lavage fluid of wild-type mice exposed to cigarette smoke but not in ECSOD-overexpressing or NOX-deficient mice. These data suggest that the mechanisms underlying the host defense against cigarette smoke-induced oxidative damage and subsequent development of COPD may include endogenous oxidases and antioxidant enzymes.

Lung EC-SOD overexpression attenuates hypoxic induction of Egr-1 and chronic hypoxic pulmonary vascular remodeling

Although production of reactive oxygen species (ROS) such as superoxide (O(2)(.-)) has been implicated in chronic hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodeling, the transcription factors and gene targets through which ROS exert their effects have not been completely identified. We used mice overexpressing the extracellular antioxidant enzyme extracellular superoxide dismutase (EC-SOD TG) to test the hypothesis that O(2)(.-) generated in the extracellular compartment under hypoxic conditions contributes to PH through the induction of the transcription factor, early growth response-1 (Egr-1), and its downstream gene target, tissue factor (TF). We found that chronic hypoxia decreased lung EC-SOD activity and protein expression in wild-type mice, but that EC-SOD activity remained five to seven times higher in EC-SOD TG mice under hypoxic conditions. EC-SOD overexpression attenuated chronic hypoxic PH, and vascular remodeling, measured by right ventricular systolic pressures, proliferation of cells in the vessel wall, muscularization of small pulmonary vessels, and collagen deposition. EC-SOD overexpression also prevented the early hypoxia-dependent upregulation of the redox-sensitive transcription factor Egr-1 and the procoagulant protein TF. These data provide the first evidence that EC-SOD activity is disrupted in chronic hypoxia, and increased EC-SOD activity can attenuate chronic hypoxic PH by limiting the hypoxic upregulation of redox-sensitive genes.

ECSOD inhibits PMN Chemotaxis by Preventing Oxidative Fragmentation of Collagen

Previous studies have found that the antioxidant enzyme extracellular superoxide dismutase (ECSOD) can inhibit neutrophillic (PMN) inflammation in a number of lung injury models. Other studies have determined that type I collagen and collagen fragments are potent chemoattractants and activators of PMNs. Notably, ECSOD binds to type I collagen and protects it from oxidative fragmentation. We therefore hypothesized that oxidatively generated fragments of collagen may enhance PMN chemotaxis and that ECSOD would inhibit this effect by preventing oxidative collagen fragmentation. To test this hypothesis, PMN chemotaxis was monitored using a modified Boyden chamber in which PMNs placed in the upper chamber of a transwell plate are separated from a type I collagen-coated lower chamber by a porous membrane. Generation of oxidants in the lower, collagen-coated chamber increased the chemotaxis of PMNs through the transwell membranes. This chemotatic response was neutralized when oxidants were generated in the presence of ECSOD. Pretreatment of PMNs with blocking antibodies to two collagen receptors, discoidin domain receptor 1 (DDR1) and the integrin 1 receptor, inhibited chemotaxis of PMNs across the membrane in response to oxidatively fragmented collagen. In summary, these studies demonstrate that oxidative collagen fragmentation induces PMN chemotaxis and that this response can be inhibited in the presence of the antioxidant enzyme ECSOD. This effect may constitute a mechanism by which ECSOD mediates inflammation in vivo.

Effect of angiotensin II type 1 receptor antagonism on endothelial function: role of bradykinin and nitric oxide

The important role of the endothelium for regulation of vascular tone, growth, inflammatory response, coagulation and thrombocyte adhesion has now been recognized. Endothelial function has largely been assessed as endothelium-dependent vasodilation, assuming that endothelium-dependent vasomotion represents a surrogate marker for other important endothelial functions. An important rational for this approach has been the observation that both endothelium-dependent vasomotion and other protective endothelial functions are at least partially mediated by nitric oxide. Accumulating clinical studies have now demonstrated a close and independent association of impaired endothelium-dependent vasodilation with cardiovascular events and prognosis. These findings have stimulated interest in treatment options to improve endothelial function in patients at high cardiovascular risk. This article describes recent insights into endothelial effects of both angiotensin-converting enzyme (ACE) inhibition and angiotensin II type 1 (AT1) receptor blockade, which have both been shown to improve endothelial function (i.e. by increasing endothelial nitric oxide availability via bradykinin-dependent endothelial nitric oxide release). ACE has a high affinity for bradykinin and degrades the peptide, so ACE inhibition may increase bradykinin-dependent effects by preventing bradykinin degradation. Interestingly, AT1-receptor blockade appears to stimulate the bradykinin-nitric oxide pathway by increased angiotensin II type 2 receptor activation. Moreover, both treatment strategies prevent increased inactivation of endothelial nitric oxide by oxygen radicals, by reducing AT1-receptor-dependent activation of the oxidant enzyme NADPH oxidase and increasing the activity of the vascular antioxidant enzyme extracellular superoxide dismutase. These beneficial effects of ACE inhibition and AT1-receptor blockade are likely to contribute to their effects on cardiovascular events.

Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase

Asbestosis is a chronic form of interstitial lung disease characterized by inflammation and fibrosis that results from the inhalation of asbestos fibers. Although the pathogenesis of asbestosis is poorly understood, reactive oxygen species may mediate the progression of this disease. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) can protect the lung against a variety of insults; however, its role in asbestosis is unknown. To determine if EC-SOD plays a direct role in protecting the lung from asbestos-induced injury, intratracheal injections of crocidolite were given to wild-type and ec-sod-null mice. Bronchoalveolar lavage fluid (BALF) from asbestos-treated ec-sod-null mice at 24 h, 14 days, or 28 days posttreatment showed increased inflammation and total BALF protein content compared to that of wild-type mice. In addition, lungs from ec-sod-null mice showed increased hydroxyproline content compared to those of wild-type mice, indicating a greater fibrotic response. Finally, lungs from ec-sod-null mice showed greater oxidative damage, as assessed by nitrotyrosine content compared to those of their wild-type counterparts. These results indicate that depletion of EC-SOD from the lung increases oxidative stress and injury in response to asbestos.

Oxidant stress—a major cause of reduced endothelial nitric oxide availability in cardiovascular disease

Common conditions predisposing to atherosclerosis, such as dyslipidemia, hypertension, diabetes, and smoking, are associated with increased vascular production of reactive oxygen species (ROS). One of the major consequences of increased vascular ROS production is a reduction of endothelial nitric oxide (NO) bioavailability. Importantly, endothelial NO not only produces endothelium-dependent vasodilation but also has potent antiatherogenic properties, including inhibition of platelet aggregation and adhesion molecule expression. This concept has been supported by several recent clinical studies, suggesting a close association of impaired endothelium-dependent vasomotion and clinical cardiovascular events. Increased vascular ROS production reduces endothelial NO not only by direct inactivation, but also as a consequence of increased oxidation of tetrahydrobiopterin and inhibition of dimethylarginine dimethylaminohydrolase. This may in part explain the profound impact of increased endothelial oxidant stress on endothelial NO bioactivity. An increased activity of NAD(P)H oxidase, a major vascular oxidant enzyme system, has been observed in both experimental atherosclerosis and human coronary disease and likely represents an important source of ROS. Moreover, NAD(P)H oxidase has been shown to cause endothelial NO synthase “uncoupling” and to promote xanthine oxidase-dependent superoxide production. In addition, in human coronary disease a reduced vascular activity of the antioxidant enzyme extracellular superoxide dismutase has been observed. Notably, cardiovascular treatment strategies such as statin, angiotensin-converting enzyme (ACE) inhibitor, and angiotensin I receptor blocker treatment may exert potent “antioxidant” effects by reducing NAD(P)H oxidase and increasing extracellular superoxide dismutase activity, leading to improved endothelial function. Restoring endothelial function has become an attractive therapeutic target, given accumulating observations supporting a prognostic role of endothelial dysfunction.

Extracellular superoxide dismutase

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.

Extracellular Superoxide Dismutase (EC-SOD) Binds to Type I Collagen and Protects Against Oxidative Fragmentation

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is mainly found in the extracellular matrix of tissues. EC-SOD participates in the detoxification of reactive oxygen species by catalyzing the dismutation of superoxide radicals. The tissue distribution of the enzyme is particularly important because of the reactive nature of its substrate, and it is likely essential that EC-SOD is positioned at the site of superoxide production to prevent adventitious oxidation. EC-SOD contains a C-terminal heparin-binding region thought to be important for modulating its distribution in the extracellular matrix. This paper demonstrates that, in addition to binding heparin, EC-SOD specifically binds to type I collagen with a dissociation constant (K(d)) of 200 nm. The heparin-binding region was found to mediate the interaction with collagen. Notably, the bound EC-SOD significantly protects type I collagen from oxidative fragmentation. This expands the known repertoire of EC-SOD binding partners and may play an important physiological role in preventing oxidative fragmentation of collagen during oxidative stress.

Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.