Secretion of extracellular superoxide dismutase in neonatal lungs

Extracellular superoxide dismutase (EC-SOD), the only known enzymatic scavenger of extracellular superoxide, may modulate reactions of nitric oxide (NO) in the lungs by preventing reactions between superoxide and NO. The regulation of EC-SOD has not been examined in developing lungs. We hypothesize that EC-SOD plays a pivotal role in the response to increased oxygen tension and NO in the neonatal lung. This study characterizes rabbit EC-SOD and investigates the developmental regulation of EC-SOD activity, protein expression, and localization. Purified rabbit EC-SOD was found to have several unique biochemical attributes distinct from EC-SOD in other species. Rabbit lung EC-SOD contains predominantly uncleaved subunits that do not form disulfide-linked dimers. The lack of intersubunit disulfide bonds may contribute to the decreased heparin affinity and lower EC-SOD content in rabbit lung. EC-SOD activity in rabbit lungs is low before birth and increases soon after gestation. In addition, the enzyme is localized intracellularly in preterm and term rabbit lungs. Secretion of active EC-SOD into the extracellular compartment increases with age. The changes in EC-SOD localization and activity have implications for the neonatal pulmonary response to oxidative stress and the biological activity of NO at birth.     

Neonatal calves develop airflow limitation due to chronic hypobaric hypoxia

Neonates and infants presenting with pulmonary hypertension and chronic hypoxia often exhibit airway obstruction. To investigate this association, we utilized a system in which neonatal calves are exposed to chronic hypobaric hypoxia and develop severe pulmonary hypertension. For the present study, one of each pair of six age-matched pairs of neonatal calves was continuously exposed to hypobaric hypoxia at 4,500 m (CH); the other remained at 1,500 m. At 2 wk of age, mean pulmonary arterial pressure (MPAP), dynamic lung compliance (Cdyn), resistance (RL), and static respiratory system compliance (Crs) were measured at 4,500 m in both CH and control calves exposed acutely to hypoxia (C). These measurements were repeated after cumulative administrations of nebulized methacholine (MCh). Tissues were removed for histological examination and assessment of bronchial ring contractility to MCh and KCl. After 2 wk of hypobaric hypoxia, MPAP (C 35 +/- 1.7 vs. CH 120 +/- 7 mmHg, P less than 0.001) and RL (C 2.64 +/- 0.16 vs CH 4.99 +/- 0.47 cmH2O.l-1s, P less than 0.001) increased. Cdyn (C 0.100 +/- 0.01 vs. CH 0.082 +/- 0.007 l/cmH2O) and Crs (CH 0.46 +/- 0.003 vs. C 0.59 +/- 0.009 l/cmH2O) were not significantly different. Compared with airways of C calves, airways of CH animals did not exhibit in vivo or in vitro MCh hyperresponsiveness; however, in vitro contractility to KCl of airways from CH animals was significantly increased. Histologically, airways from the CH calves showed increases in airway fibrous tissue and smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular superoxide dismutase overexpression can reverse the course of hypoxia-induced pulmonary hypertension

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.     

Decreased pulmonary extracellular superoxide dismutase during systemic inflammation

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

Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.     

Expression profiles of extracellular superoxide dismutase during mouse organogenesis

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.     

Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.     

缺氧对家兔肺泡表面活性物质中各磷脂组分的影响

肺泡表面活性物质中各磷脂组分对缺氧的反应是不同的。急性缺氧时,肺泡冲洗液中溶血卵磷脂、神经鞘磷脂、磷脂酰乙醇胺及磷脂酰甘油均下降(p<0.05);间断适应性缺氧后,基本回复到缺氧前水平。而急性缺氧时,肺组织中除磷脂酰乙醇胺变化不明显外,其它各成分均有明显增加(p<0.05)。间断缺氧时,肺组织中各脂质成分持续下降。该变化可能与肺组织中ATP等能量物质在缺氧时代谢异常有关。从肺泡冲洗液的脂质分析结果来看,适当地以间断减压作缺氧适应,能有效地解除急性缺氧对肺泡表面磷脂含量的抑制作用。     

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.     

Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 +/- 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 +/- 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 +/- 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.     

Hypoxia-induced increase of endostatin in murine aorta and lung

In the lung, hypoxia induces pulmonary hypertension caused by vasoconstriction and vascular remodeling. Additionally, hypoxia is an inducer of angiogenesis, which is assumed to counteract pulmonary hypertension. We asked whether the anti-angiogenic factor endostatin—a cleavage product of collagen XVIII—participates in the vascular alterations induced by hypoxia. By employing Western blotting of tissue extracts of murine brain, liver and heart an endostatin fragment of 22 kDa was detectable, whereas in lung and aorta additional bands of 24 and 26 kDa were found. The amount of these larger fragments was increased in tissues obtained from mice housed for 4 days or 3 weeks at hypobaric hypoxia. By immunohistochemistry endostatin was detected in association with elastic fibers and in close neighborhood to smooth muscle cells of intrapulmonary vessels and the aorta. In the lung, the activity of matrix metalloproteinases (MMP) known to generate endostatin by cleavage of collagen XVIII was increased (MMP-2) and decreased (proMMP-9), respectively, by hypoxia. Elevated amounts of endostatin within the aortic wall of mice exposed to hypobaric hypoxia may stabilize the vascular wall by inhibition of microvascular sprouting. The surprising finding of increased endostatin in the lung presumably contributes to the development of pulmonary hypertension by reduction of angiogenesis.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Purification and characterization of extracellular superoxide dismutase in mouse lung

Extracellular superoxide dismutase (EC-SOD) is the major isozyme of SOD in arteries, but is also abundant in lungs. In particular, mouse lungs contain large amounts of EC-SOD compared to lungs in other mammals. This suggests that EC-SOD may have an amplified function in the mouse lung. This study describes the purification and characterization of mouse EC-SOD as well as its localization in mouse lung. Mouse EC-SOD exists primarily as a homotetramer composed of a pair of dimers linked through disulfide bonds present in the heparin-binding domains of each subunit. In addition, mouse EC-SOD can exist in active multimeric forms. We developed and utilized a polyclonal antibody to mouse EC-SOD to immunolocalize EC-SOD in mouse lung. EC-SOD labeling is strongest in the matrix of vessels, airways, and alveolar septa. This localization suggests that EC-SOD may have important functions in pulmonary biology, perhaps in the modulation of nitric oxide-dependent responses.     

Carbon monoxide promotes hypoxic pulmonary vascular remodeling

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.     

Role of barometric pressure in pulmonary fluid balance and oxygen transport

To examine the role of barometric pressure in high-altitude pulmonary edema, we randomly exposed five unanesthetized chronically instrumented sheep with lung lymph fistulas in a decompression chamber to each of three separate conditions: hypobaric hypoxia, normobaric hypoxia, and normoxic hypobaria. A combination of slow decompression and/or simultaneous adjustment of inspired PO2 provided three successive stages of simulated altitudes of 2,600, 4,600, and 6,600 m during which hemodynamics and lymph flow were monitored. Under both hypoxic conditions we noted significant and equivalent elevations in pulmonary arterial pressure (Ppa), cardiac output, and heart rate, with left atrial and systemic pressures remaining fairly constant. Normoxic hypobaria was also accompanied by a smaller but significant rise in Ppa. Lymph flow increased to a highly significant maximum of 73% above base line, accompanied by a slight but significant decrease in lung lymph-to-plasma protein ratio, only under conditions of combined hypobaric hypoxia but not under equivalent degrees of alveolar hypoxia or hypobaria alone. Arterial hypoxemia was noted under all three conditions, with arterial PO2 being uniformly lower under hypobaric conditions than when identical amounts of inspired PO2 were delivered at normal atmospheric pressure. We therefore hypothesize that alveolar pressure significantly alters the Starling forces governing transcapillary fluid flux in the lung and may affect the alveolar-arterial gradient for O2 as well.     

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.