Extracellular Superoxide Dismutase Protects Lung Development in Hyperoxia-exposed Newborn Mice
Departments of Pediatrics, Medicine, Anesthesiology, Cell Biology, Duke University Medical Center, Durham, North Carolinaw'(y\1, 百拇医药
ABSTRACTw'(y\1, 百拇医药
TOPw'(y\1, 百拇医药
ABSTRACTw'(y\1, 百拇医药
METHODSw'(y\1, 百拇医药
RESULTSw'(y\1, 百拇医药
DISCUSSIONw'(y\1, 百拇医药
REFERENCESw'(y\1, 百拇医药
We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.
Key Words: bronchopulmonary dysplasia • superoxide dismutase • hyperoxia • newborn infant • transgenic micec&8n, 百拇医药
In extreme prematurity, respiratory failure due to deficient alveolar development and surfactant production could be complicated by diminished antioxidant stores and enzymatic antioxidant inducibility (1). Inflammatory responses to perinatal infection or lung injury cause further cellular damage by increasing oxidant and proteolytic reactions. Bronchopulmonary dysplasia (BPD) remains a common complication of extreme prematurity, despite advances in surfactant therapy and intensive care. It has recently been characterized as a failure of complete alveolar development (2). Proposed mechanisms include oxidant damage to biomolecules that lead to indirect effects on cell proliferation. Hyperoxia impairs alveolar formation (3), and the relative deficiency of antioxidant defenses may render 21% FIO2 "hyperoxic" in premature newborns (4). Inflammation contributes by effects of leukocyte-mediated proteolytic enzymatic cellular damage and by releasing oxygen radicals produced during the respiratory burst that further oxidize biomolecules (5). Oxidant injury and inflammation are thought to be dominant mechanisms in the pathogenesis of BPD (6).
Premature newborns are relatively deficient in stores of antioxidant vitamins A and E (7). Although relative pulmonary antioxidant enzyme immaturity has not been corroborated in humans, cord blood antioxidant levels decline with decreasing gestational age (8). There is ample evidence that clinically encountered oxidant stress in premature newborns is accompanied by lung protein and lipid oxidation (9, 10). The rationale for supplementing antioxidant capacity is also supported by studies that supplied either iron chelators (deferoxamine) or iron-binding proteins (transferrin) and demonstrated reduced oxidant stress and improved lung growth, as recently reviewed by Frank and Sosenko (7). At present, antioxidant vitamin prophylaxis does not prevent BPD in a baboon model, (11) and clinical trials of antioxidant vitamin treatment (12) or antioxidant enzyme supplementation (13) have had only modest success or have been ineffective at preventing BPD.mr{*%, 百拇医药
Failure of exogenous antioxidant enzyme prevention of BPD may be due to nonspecific effects or ineffective dose delivery to vulnerable lung compartments (14, 15). Overexpression of manganese superoxide dismutase (SOD) conferred superior survival in hyperoxia-exposed transgenic adult mice (16).
We recently demonstrated protection from hyperoxia in human extracellular SOD (hEC-SOD) transgenic hyperoxia-exposed adult mice that typically express two- to threefold increases in total EC-SOD activity over wild-type littermates (17). In contrast, EC-SOD knockout mice had increased mortality and lung injury when exposed to hyperoxia (18). EC-SOD may confer the particular advantage of targeting the vulnerable extracellular milieu of the lung epithelium, potentially providing superior protection against oxidant-mediated cellular injury, as recently suggested by Berrington and colleagues (14). This may permit normal proliferation and differentiation in newborns, which is known to be inhibited by hyperoxia (19).h&ahdq, 百拇医药
To determine whether sustained expression of EC-SOD would promote normal lung development during severe oxidative stress, we evaluated the effect of transgenic overexpression of hEC-SOD in a mouse model of hyperoxia-induced chronic lung disease. In this model, newborn mice are exposed to FIO2 = 0.95 at birth for 1 week and then recovered at FIO2 = 0.6 for two more weeks to induce an oxidative and inflammatory insult followed by more indolent oxidative stress.
METHODSeo, 百拇医药
TOPeo, 百拇医药
ABSTRACTeo, 百拇医药
METHODSeo, 百拇医药
RESULTSeo, 百拇医药
DISCUSSIONeo, 百拇医药
REFERENCESeo, 百拇医药
We used B6C3 transgenic mice, (17) with the human SP-C promoter targeting human EC-SOD expression mainly in alveolar epithelium (20). Experiments were performed according to institutional guidelines for animal care and use at Duke University Medical Center.eo, 百拇医药
Acute Hyperoxia Exposureeo, 百拇医药
Newborn mice (wild-type and transgenic) were randomly assigned to air and oxygen exposure (FIO2 = 95% for 7 days) beginning at birth. Nursing dams were alternated between air and oxygen-exposed litters (21). Animals were killed with 150 mg/kg intraperitoneal sodium pentobarbital after exposure. Genotype analysis was performed using polymerase chain reaction (17). Body weights, survival, and lung weights were recorded.
EC-SOD Expression by Activitym, 百拇医药
Tissues from three or four mice in each treatment group were weighed and homogenized in a buffer containing an anti-protease cocktail (see details in the online supplement). Supernatants obtained after centrifugation were pooled and passed over a concanavalin-A Sepharose column to isolate EC-SOD, and activity was measured three or four times by inhibition of xanthine/xanthine oxidase-mediated cytochrome c reduction, as described previously (17).m, 百拇医药
EC-SOD Expression by Immunoblotm, 百拇医药
Lungs were homogenized, and 20 µg total lung protein from each animal was analyzed by western blotting, detected with anti-mouse EC-SOD or anti-human EC-SOD as described in the online supplement.m, 百拇医药
EC-SOD Expression by Immunohistochemistrym, 百拇医药
Lungs from five mice in each treatment group were fixed as described previously (22). Sections from inflation-fixed lung were immunostained for hEC-SOD. We evaluated the histochemical signal as described previously using rabbit anti-human EC-SOD 1:3,000 and detected them with biotinylated goat anti-rabbit 1:4,000 (23). Preimmune rabbit serum was used as a control for the EC-SOD immunostaining.
Total and Oxidized Glutathionem9-{, 百拇医药
Total and oxidized glutathione was measured in lung homogenates from a minimum of four pups/treatment group by high-performance liquid chromatography according to a procedure slightly modified from Paroni and coworkers (24).m9-{, 百拇医药
Bronchoalveolar Lavagem9-{, 百拇医药
Five wild-type and five trangenic pups/treatment group underwent bronchoalveolar lavage (BAL) to determine cell counts using 0.5 ml 0.9% sodium chloride, 1 mM ethylenediamine tetraacetic acid, and repeated four times before pooling (25). Total cell counts from BAL fluid were counted in a hemacytometer, then cells were cytocentrifuged, and Wright stained. At least 300 cells were counted to obtain a differential leukocyte cell count.m9-{, 百拇医药
Myeloperoxidasem9-{, 百拇医药
Myeloperoxidase activity was measured spectrophotometrically in whole lung homogenates from five animals/treatment group by reaction with o-dianisidine dye using a microplate assay described previously (21, 22).
Chronic Hyperoxia Exposure(, 百拇医药
In other animals, after 7 days air or hyperoxia, the FIO2 in the hyperoxia groups was reduced from 95 to 60% until age 21 days, at which time all pups were killed and lungs inflation-fixed and sectioned as described previously. Survival, body weights, and lung weights were measured. Five animals/treatment group were evaluated morphometrically to determine alveolar surface density and alveolar volume density as estimates of surface area and alveolar number, as described previously and in the online supplement (26). Activities of EC-SOD were measured in lung homogenates pooled from four animals/treatment group to achieve adequate detection. Whole lung myeloperoxidase measurements were made in five animals/treatment group as in the acute hyperoxia studies referred to previously.(, 百拇医药
Statistical Analysis(, 百拇医药
Groups were compared using analysis of variance, and a post hoc Tukey–Kramer test was performed to determine statistical differences. We accepted p values less than 0.05 as significant, assuming an error = 0.05 and ß error = 0.10. Analysis was performed using JMP Software version 3.2 (SAS, Cary, NC).
RESULTSyx;, 百拇医药
TOPyx;, 百拇医药
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METHODSyx;, 百拇医药
RESULTSyx;, 百拇医药
DISCUSSIONyx;, 百拇医药
REFERENCESyx;, 百拇医药
Effects of Acute Hyperoxia on Newborn Mouse Body and Lung Weightsyx;, 百拇医药
There was no mortality in the air-exposed groups. At 7 days, 95% oxygen exposure resulted in 17% mortality for wild-type and 15% for transgenic mice (p = 0.50, not significant) and decreased body weight in both genotypes: wild-type 2.9 ± 0.6 g, n = 56, versus transgenic 2.8 ± 0.3 g, n = 62 (mean ± SEM). Hyperoxia exposure decreased lung weight: air, 76.7 ± 8.0 versus hyperoxia: 62.0 + 1.4, p value less than 0.05, and n = 9/group. Genotype had no effect on lung weight.yx;, 百拇医药
EC-SOD expression.yx;, 百拇医药
We found that EC-SOD activity in air-exposed transgenic mice was more than double the activity compared with wild-type littermates, as shown in . This increase was further induced by hyperoxia at 7 days in transgenic mice. Immunoblotting showed that human EC-SOD was detected only in the transgenic mice, but mouse EC-SOD was detected in both wild-type and transgenic mice. Hyperoxia had no effect on either human or mouse EC-SOD protein expression measured by immunoblot . In the transgenic newborn mice, hEC-SOD immunohistochemical expression was primarily located in alveolar epithelium and also in bronchiolar epithelium . No hEC-SOD immunostaining was seen in wild-type littermates.
fig.ommittedj!s9|:, http://www.100md.com
Figure 1. (A) Total (mouse + human) EC-SOD activity in hyperoxia-exposed wild-type and transgenic newborn mice at 7 days. Mean + SD of three or four replicate measurements from pooled samples, n = 4 per group, *p value less than 0.05 versus wild-type, p value less than 0.05 versus EC-SOD air. (B) Immunoblot of total lung protein detected with anti-human EC-SOD, 20 µg protein/lane (upper) and anti-mouse EC-SOD, 10 µg protein/lane (lower) in wild-type versus transgenic, air versus 95% oxygen x 7 days.j!s9|:, http://www.100md.com
fig.ommittedj!s9|:, http://www.100md.com
Figure 2. Immunolocalization of human EC-SOD (does not recognize mouse EC-SOD) in newborn wild-type (A) and transgenic (B) mouse lung, air-exposed, 200x magnification.j!s9|:, http://www.100md.com
Glutathione.j!s9|:, http://www.100md.com
Total glutathione in lung homogenates was increased by hyperoxia exposure at 7 days but less so in EC-SOD transgenic mice. Lung homogenates from mice exposed to hyperoxia for 7 days had elevated oxidized glutathione compared with air-exposed mice. The elevation in the EC-SOD transgenic mice was slightly less than in the wild-type mice. Hyperoxia-exposed transgenic mice had significantly lower levels of oxidized and reduced glutathione than hyperoxia-exposed wild-type littermates .
fig.ommitted8l)0-u1, 百拇医药
Figure 3. Effect of hEC-SOD overexpression on oxidized, reduced, and total glutathione in lung homogenates from hyperoxia-exposed newborn mice at 7 days. n = 5 per group. Mean values, *p = 0.004, **p = 0.036, ***p = 0.013, hyperoxia-exposed transgenic versus hyperoxia-exposed wild-type.8l)0-u1, 百拇医药
Inflammation.8l)0-u1, 百拇医药
Transgenic mice exposed to hyperoxia for 7 days showed a qualitative reduction in the inflammatory cell infiltrate and preservation of septal integrity . Hyperoxia increased BAL leukocyte accumulation in both wild-type and transgenic mice, but transgenic mice had significantly reduced BAL leukocytes compared with wild-type littermates. Transgenic hEC-SOD expression also prevented accumulation of MPO activity in whole lung homogenates from hyperoxia-exposed mice compared with wild-type littermates.8l)0-u1, 百拇医药
fig.ommitted8l)0-u1, 百拇医药
Figure 4. Effect of hEC-SOD overexpression on inflammation in hyperoxia-exposed newborn mouse lung at 7 days. Representative photomicrographs illustrate the effects on cellular influx, 200x magnification (upper panels), BAL leukocytes and polymorphonuclear leukocytes (lower left), and tissue myeloperoxidase (lower right). n = 5 per group. Mean + SEM, *p value less than 0.05 versus air-exposure, p value less than 0.05 versus wild-type.
Chronic Hyperoxia@}^)g-[, 百拇医药
Survival, body and lung weights.@}^)g-[, 百拇医药
Chronic hyperoxia impaired growth: hyperoxia, 8.9 ± 0.4 g, n = 15, versus air, 11.4 ± 0.7 g, n = 22, p value less than 0.001. Genotype had no effect on body weight or lung weight at 21 days in either air or hyperoxia exposure. After 21 days, lung wet weights were similar in air and hyperoxia exposed groups, hyperoxia, 97 ± 3 mg, versus air 89 ± 5mg, n = 14/group p = 0.16. There were no differences in late mortality in wild-type versus transgenic hyperoxia-exposed mice.@}^)g-[, 百拇医药
Alveolar development.@}^)g-[, 百拇医药
There were no differences in surface density or volume density between 21-day air-exposed wild-type and transgenic mice. Hyperoxia exposure for 21 days significantly reduced alveolar surface density and alveolar volume density in wild-type mice but not in EC-SOD transgenic mice .@}^)g-[, 百拇医药
fig.ommitted@}^)g-[, 百拇医药
Figure 5. Effect of hEC-SOD overexpression on alveolar development after 21 days of air or hyperoxia. Surface density (A) and volume density (B). n = 5 per group. Mean + SEM, *p value less than 0.05 versus transgenic, hyperoxia-exposed.
Inflammation.!0, 百拇医药
Tissue myeloperoxidase accumulation was similar among all groups at Day 21 . There was a trend toward decreased myeloperoxidase accumulation in the hEC-SOD transgenic animals in both treatment groups, but this did not reach statistical significance.!0, 百拇医药
fig.ommitted!0, 百拇医药
Figure 6. Effect of hEC-SOD overexpression on lung myeloperoxidase accumulation in hyperoxia-exposed newborn mouse at 21 days. n = 5 per group. Mean + SEM, *p value less than 0.05 versus transgenic, hyperoxia-exposed.!0, 百拇医药
EC-SOD activity.!0, 百拇医药
EC-SOD transgenic mice maintained elevated total lung EC-SOD activity at 21 days. Transgenic animals had more than threefold the activity levels of those observed in wild-type littermates, as in the 7-day exposed newborns . Hyperoxia-exposure induced an additional increase in total EC-SOD activity compared with air-exposed transgenic animals. Hyperoxia had no effect on EC-SOD activity in wild-type littermates.
fig.ommittedt|, http://www.100md.com
Figure 7. EC-SOD activity in mouse lung at 21 days, air versus chronic hyperoxia, wild-type versus transgenic. Mean + SD of three replicate measurements from pooled samples, n = 4 per group, *p value less than 0.05 versus wild-type, p value less than 0.05 versus EC-SOD air.t|, http://www.100md.com
DISCUSSIONt|, http://www.100md.com
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ABSTRACTt|, http://www.100md.com
METHODSt|, http://www.100md.com
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DISCUSSIONt|, http://www.100md.com
REFERENCESt|, http://www.100md.com
Overexpression of human EC-SOD in hyperoxia-exposed adult mice confers improved survival and decreased inflammation (17). In our model of chronic lung disease, we sought to use a sublethal injury that shares some features of human chronic lung disease, namely oxidant stress, inflammation, and impaired alveolar development (16). We found that overexpression of human EC-SOD targeted to pulmonary epithelium partly prevented glutathione oxidation, and reduced hyperoxia-induced interstitial and airway inflammation at 7 days. Later alveolar development—as determined by alveolar surface and volume density—was preserved in hEC-SOD transgenic mice exposed to hyperoxia for 21 days.
EC-SOD expression may confer particular protection against oxidant stress. Mutant mice lacking EC-SOD are more susceptible to hyperoxia-induced injury and inflammation (18). Transgenic mice overexpressing mitochondrial (manganese) SOD showed improved survival and attenuation of acute injury (16). Hyperoxia-exposure studies in transgenic mice overexpressing cytosolic (copper–zinc) SOD conferred similar benefits (27). Because EC-SOD is a secreted protein, it may dismutate superoxide in vulnerable lung compartments relatively inaccessible to intracellular antioxidants, such as the surface of alveolar epithelium or the alveolar hypophase (28).r., 百拇医药
Inflammation may exacerbate oxidant stress and thus contribute to propagation of lung injury, and it may impede repair and cell proliferation. Our previous studies in hyperoxia-exposed adult EC-SOD overexpressing mice showed attenuation of inflammatory cytokines and adhesion molecules (17). We have recently shown that blockade of neutrophil influx protects against adverse hyperoxia effects on lung development in newborn rats (26). The preservation of alveolar development we observed at 21 days in the present studies may be partly attributable to the early reduction of inflammation we observed as reduced BAL fluid leukocyte counts and tissue myeloperoxidase in transgenic mice at 7 days.
Overexpression of EC-SOD preserved lung development during oxidative stress, evident as partly preserved alveolar surface and volume density. This would indicate that secondary septation is likely preserved because decreased septation would result in lower surface density. There were no differences in surface density between air-exposed newborn wild-type and transgenic mice. This suggests that EC-SOD transgenic animals do not have constitutively superior alveolar development in the absence of oxidative stress.2qw, 百拇医药
The beneficial effects of EC-SOD overexpression may be mediated indirectly by alterations in ROS signaling of cellular processes. Hydrogen peroxide, produced spontaneously or by SOD by single electron reduction of superoxide, has been increasingly recognized as a mediator of in vitro apoptosis and may alter cell proliferation (29). Reduction of hydrogen peroxide by exogenous catalase in vitro can abrogate the effect of cellular mitogens, as recently reviewed by Jankov and coworkers(30). Potential effects of SOD on hydrogen peroxide–mediated signaling would likely depend greatly on the timing, concentration, and location of hydrogen peroxide.
Hyperoxia induced even higher levels of total EC-SOD activity in the transgenic animals at 7 and 21 days. The transferred hEC-SOD gene is under control of the human SP-C promoter in the transgenic mice. It is known that hyperoxia induces increased SP-C messenger RNA in mice, and we speculate that similar mechanisms may be enhancing the human SP-C promoter–controlled transgenic EC-SOD overexpression (31, 32). In contrast, human EC-SOD protein levels measured by immunoblot did not appear to be induced by hyperoxia, but this was not strictly quantified. Cellular protein processing in the transgenic mice may alter the EC-SOD activity without changing the total lung EC-SOD accumulation. The wild-type littermates showed no hyperoxia-induced changes in either EC-SOD activity or mouse EC-SOD accumulation in whole lung.g, http://www.100md.com
We conclude that overexpression of hEC-SOD in neonatal mice protects against hyperoxia-induced inflammation at 7 days and impairment of lung development at 21 days. Although the specific mechanism is still unclear, it likely includes preservation of reduced glutathione—important for lung oxidant–antioxidant equilibrium—and attenuation of inflammation that may cause cell death and alveolar destruction. Newborn mice exposed to hyperoxia for 21 days showed maintenance of both alveolar volume and surface density. We speculate that correctly targeted SOD overexpression will prevent adverse effects of oxidant stress on lung development and may protect lung development in newborns at risk to develop BPD.
Acknowledgments+'3, 百拇医药
The authors gratefully acknowledge the technical assistance of Mary Whorton and Kathryn Auten.+'3, 百拇医药
REFERENCES+'3, 百拇医药
TOP+'3, 百拇医药
ABSTRACT+'3, 百拇医药
METHODS+'3, 百拇医药
RESULTS+'3, 百拇医药
DISCUSSION+'3, 百拇医药
REFERENCES+'3, 百拇医药
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ABSTRACTw'(y\1, 百拇医药
TOPw'(y\1, 百拇医药
ABSTRACTw'(y\1, 百拇医药
METHODSw'(y\1, 百拇医药
RESULTSw'(y\1, 百拇医药
DISCUSSIONw'(y\1, 百拇医药
REFERENCESw'(y\1, 百拇医药
We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.
Key Words: bronchopulmonary dysplasia • superoxide dismutase • hyperoxia • newborn infant • transgenic micec&8n, 百拇医药
In extreme prematurity, respiratory failure due to deficient alveolar development and surfactant production could be complicated by diminished antioxidant stores and enzymatic antioxidant inducibility (1). Inflammatory responses to perinatal infection or lung injury cause further cellular damage by increasing oxidant and proteolytic reactions. Bronchopulmonary dysplasia (BPD) remains a common complication of extreme prematurity, despite advances in surfactant therapy and intensive care. It has recently been characterized as a failure of complete alveolar development (2). Proposed mechanisms include oxidant damage to biomolecules that lead to indirect effects on cell proliferation. Hyperoxia impairs alveolar formation (3), and the relative deficiency of antioxidant defenses may render 21% FIO2 "hyperoxic" in premature newborns (4). Inflammation contributes by effects of leukocyte-mediated proteolytic enzymatic cellular damage and by releasing oxygen radicals produced during the respiratory burst that further oxidize biomolecules (5). Oxidant injury and inflammation are thought to be dominant mechanisms in the pathogenesis of BPD (6).
Premature newborns are relatively deficient in stores of antioxidant vitamins A and E (7). Although relative pulmonary antioxidant enzyme immaturity has not been corroborated in humans, cord blood antioxidant levels decline with decreasing gestational age (8). There is ample evidence that clinically encountered oxidant stress in premature newborns is accompanied by lung protein and lipid oxidation (9, 10). The rationale for supplementing antioxidant capacity is also supported by studies that supplied either iron chelators (deferoxamine) or iron-binding proteins (transferrin) and demonstrated reduced oxidant stress and improved lung growth, as recently reviewed by Frank and Sosenko (7). At present, antioxidant vitamin prophylaxis does not prevent BPD in a baboon model, (11) and clinical trials of antioxidant vitamin treatment (12) or antioxidant enzyme supplementation (13) have had only modest success or have been ineffective at preventing BPD.mr{*%, 百拇医药
Failure of exogenous antioxidant enzyme prevention of BPD may be due to nonspecific effects or ineffective dose delivery to vulnerable lung compartments (14, 15). Overexpression of manganese superoxide dismutase (SOD) conferred superior survival in hyperoxia-exposed transgenic adult mice (16).
We recently demonstrated protection from hyperoxia in human extracellular SOD (hEC-SOD) transgenic hyperoxia-exposed adult mice that typically express two- to threefold increases in total EC-SOD activity over wild-type littermates (17). In contrast, EC-SOD knockout mice had increased mortality and lung injury when exposed to hyperoxia (18). EC-SOD may confer the particular advantage of targeting the vulnerable extracellular milieu of the lung epithelium, potentially providing superior protection against oxidant-mediated cellular injury, as recently suggested by Berrington and colleagues (14). This may permit normal proliferation and differentiation in newborns, which is known to be inhibited by hyperoxia (19).h&ahdq, 百拇医药
To determine whether sustained expression of EC-SOD would promote normal lung development during severe oxidative stress, we evaluated the effect of transgenic overexpression of hEC-SOD in a mouse model of hyperoxia-induced chronic lung disease. In this model, newborn mice are exposed to FIO2 = 0.95 at birth for 1 week and then recovered at FIO2 = 0.6 for two more weeks to induce an oxidative and inflammatory insult followed by more indolent oxidative stress.
METHODSeo, 百拇医药
TOPeo, 百拇医药
ABSTRACTeo, 百拇医药
METHODSeo, 百拇医药
RESULTSeo, 百拇医药
DISCUSSIONeo, 百拇医药
REFERENCESeo, 百拇医药
We used B6C3 transgenic mice, (17) with the human SP-C promoter targeting human EC-SOD expression mainly in alveolar epithelium (20). Experiments were performed according to institutional guidelines for animal care and use at Duke University Medical Center.eo, 百拇医药
Acute Hyperoxia Exposureeo, 百拇医药
Newborn mice (wild-type and transgenic) were randomly assigned to air and oxygen exposure (FIO2 = 95% for 7 days) beginning at birth. Nursing dams were alternated between air and oxygen-exposed litters (21). Animals were killed with 150 mg/kg intraperitoneal sodium pentobarbital after exposure. Genotype analysis was performed using polymerase chain reaction (17). Body weights, survival, and lung weights were recorded.
EC-SOD Expression by Activitym, 百拇医药
Tissues from three or four mice in each treatment group were weighed and homogenized in a buffer containing an anti-protease cocktail (see details in the online supplement). Supernatants obtained after centrifugation were pooled and passed over a concanavalin-A Sepharose column to isolate EC-SOD, and activity was measured three or four times by inhibition of xanthine/xanthine oxidase-mediated cytochrome c reduction, as described previously (17).m, 百拇医药
EC-SOD Expression by Immunoblotm, 百拇医药
Lungs were homogenized, and 20 µg total lung protein from each animal was analyzed by western blotting, detected with anti-mouse EC-SOD or anti-human EC-SOD as described in the online supplement.m, 百拇医药
EC-SOD Expression by Immunohistochemistrym, 百拇医药
Lungs from five mice in each treatment group were fixed as described previously (22). Sections from inflation-fixed lung were immunostained for hEC-SOD. We evaluated the histochemical signal as described previously using rabbit anti-human EC-SOD 1:3,000 and detected them with biotinylated goat anti-rabbit 1:4,000 (23). Preimmune rabbit serum was used as a control for the EC-SOD immunostaining.
Total and Oxidized Glutathionem9-{, 百拇医药
Total and oxidized glutathione was measured in lung homogenates from a minimum of four pups/treatment group by high-performance liquid chromatography according to a procedure slightly modified from Paroni and coworkers (24).m9-{, 百拇医药
Bronchoalveolar Lavagem9-{, 百拇医药
Five wild-type and five trangenic pups/treatment group underwent bronchoalveolar lavage (BAL) to determine cell counts using 0.5 ml 0.9% sodium chloride, 1 mM ethylenediamine tetraacetic acid, and repeated four times before pooling (25). Total cell counts from BAL fluid were counted in a hemacytometer, then cells were cytocentrifuged, and Wright stained. At least 300 cells were counted to obtain a differential leukocyte cell count.m9-{, 百拇医药
Myeloperoxidasem9-{, 百拇医药
Myeloperoxidase activity was measured spectrophotometrically in whole lung homogenates from five animals/treatment group by reaction with o-dianisidine dye using a microplate assay described previously (21, 22).
Chronic Hyperoxia Exposure(, 百拇医药
In other animals, after 7 days air or hyperoxia, the FIO2 in the hyperoxia groups was reduced from 95 to 60% until age 21 days, at which time all pups were killed and lungs inflation-fixed and sectioned as described previously. Survival, body weights, and lung weights were measured. Five animals/treatment group were evaluated morphometrically to determine alveolar surface density and alveolar volume density as estimates of surface area and alveolar number, as described previously and in the online supplement (26). Activities of EC-SOD were measured in lung homogenates pooled from four animals/treatment group to achieve adequate detection. Whole lung myeloperoxidase measurements were made in five animals/treatment group as in the acute hyperoxia studies referred to previously.(, 百拇医药
Statistical Analysis(, 百拇医药
Groups were compared using analysis of variance, and a post hoc Tukey–Kramer test was performed to determine statistical differences. We accepted p values less than 0.05 as significant, assuming an error = 0.05 and ß error = 0.10. Analysis was performed using JMP Software version 3.2 (SAS, Cary, NC).
RESULTSyx;, 百拇医药
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DISCUSSIONyx;, 百拇医药
REFERENCESyx;, 百拇医药
Effects of Acute Hyperoxia on Newborn Mouse Body and Lung Weightsyx;, 百拇医药
There was no mortality in the air-exposed groups. At 7 days, 95% oxygen exposure resulted in 17% mortality for wild-type and 15% for transgenic mice (p = 0.50, not significant) and decreased body weight in both genotypes: wild-type 2.9 ± 0.6 g, n = 56, versus transgenic 2.8 ± 0.3 g, n = 62 (mean ± SEM). Hyperoxia exposure decreased lung weight: air, 76.7 ± 8.0 versus hyperoxia: 62.0 + 1.4, p value less than 0.05, and n = 9/group. Genotype had no effect on lung weight.yx;, 百拇医药
EC-SOD expression.yx;, 百拇医药
We found that EC-SOD activity in air-exposed transgenic mice was more than double the activity compared with wild-type littermates, as shown in . This increase was further induced by hyperoxia at 7 days in transgenic mice. Immunoblotting showed that human EC-SOD was detected only in the transgenic mice, but mouse EC-SOD was detected in both wild-type and transgenic mice. Hyperoxia had no effect on either human or mouse EC-SOD protein expression measured by immunoblot . In the transgenic newborn mice, hEC-SOD immunohistochemical expression was primarily located in alveolar epithelium and also in bronchiolar epithelium . No hEC-SOD immunostaining was seen in wild-type littermates.
fig.ommittedj!s9|:, http://www.100md.com
Figure 1. (A) Total (mouse + human) EC-SOD activity in hyperoxia-exposed wild-type and transgenic newborn mice at 7 days. Mean + SD of three or four replicate measurements from pooled samples, n = 4 per group, *p value less than 0.05 versus wild-type, p value less than 0.05 versus EC-SOD air. (B) Immunoblot of total lung protein detected with anti-human EC-SOD, 20 µg protein/lane (upper) and anti-mouse EC-SOD, 10 µg protein/lane (lower) in wild-type versus transgenic, air versus 95% oxygen x 7 days.j!s9|:, http://www.100md.com
fig.ommittedj!s9|:, http://www.100md.com
Figure 2. Immunolocalization of human EC-SOD (does not recognize mouse EC-SOD) in newborn wild-type (A) and transgenic (B) mouse lung, air-exposed, 200x magnification.j!s9|:, http://www.100md.com
Glutathione.j!s9|:, http://www.100md.com
Total glutathione in lung homogenates was increased by hyperoxia exposure at 7 days but less so in EC-SOD transgenic mice. Lung homogenates from mice exposed to hyperoxia for 7 days had elevated oxidized glutathione compared with air-exposed mice. The elevation in the EC-SOD transgenic mice was slightly less than in the wild-type mice. Hyperoxia-exposed transgenic mice had significantly lower levels of oxidized and reduced glutathione than hyperoxia-exposed wild-type littermates .
fig.ommitted8l)0-u1, 百拇医药
Figure 3. Effect of hEC-SOD overexpression on oxidized, reduced, and total glutathione in lung homogenates from hyperoxia-exposed newborn mice at 7 days. n = 5 per group. Mean values, *p = 0.004, **p = 0.036, ***p = 0.013, hyperoxia-exposed transgenic versus hyperoxia-exposed wild-type.8l)0-u1, 百拇医药
Inflammation.8l)0-u1, 百拇医药
Transgenic mice exposed to hyperoxia for 7 days showed a qualitative reduction in the inflammatory cell infiltrate and preservation of septal integrity . Hyperoxia increased BAL leukocyte accumulation in both wild-type and transgenic mice, but transgenic mice had significantly reduced BAL leukocytes compared with wild-type littermates. Transgenic hEC-SOD expression also prevented accumulation of MPO activity in whole lung homogenates from hyperoxia-exposed mice compared with wild-type littermates.8l)0-u1, 百拇医药
fig.ommitted8l)0-u1, 百拇医药
Figure 4. Effect of hEC-SOD overexpression on inflammation in hyperoxia-exposed newborn mouse lung at 7 days. Representative photomicrographs illustrate the effects on cellular influx, 200x magnification (upper panels), BAL leukocytes and polymorphonuclear leukocytes (lower left), and tissue myeloperoxidase (lower right). n = 5 per group. Mean + SEM, *p value less than 0.05 versus air-exposure, p value less than 0.05 versus wild-type.
Chronic Hyperoxia@}^)g-[, 百拇医药
Survival, body and lung weights.@}^)g-[, 百拇医药
Chronic hyperoxia impaired growth: hyperoxia, 8.9 ± 0.4 g, n = 15, versus air, 11.4 ± 0.7 g, n = 22, p value less than 0.001. Genotype had no effect on body weight or lung weight at 21 days in either air or hyperoxia exposure. After 21 days, lung wet weights were similar in air and hyperoxia exposed groups, hyperoxia, 97 ± 3 mg, versus air 89 ± 5mg, n = 14/group p = 0.16. There were no differences in late mortality in wild-type versus transgenic hyperoxia-exposed mice.@}^)g-[, 百拇医药
Alveolar development.@}^)g-[, 百拇医药
There were no differences in surface density or volume density between 21-day air-exposed wild-type and transgenic mice. Hyperoxia exposure for 21 days significantly reduced alveolar surface density and alveolar volume density in wild-type mice but not in EC-SOD transgenic mice .@}^)g-[, 百拇医药
fig.ommitted@}^)g-[, 百拇医药
Figure 5. Effect of hEC-SOD overexpression on alveolar development after 21 days of air or hyperoxia. Surface density (A) and volume density (B). n = 5 per group. Mean + SEM, *p value less than 0.05 versus transgenic, hyperoxia-exposed.
Inflammation.!0, 百拇医药
Tissue myeloperoxidase accumulation was similar among all groups at Day 21 . There was a trend toward decreased myeloperoxidase accumulation in the hEC-SOD transgenic animals in both treatment groups, but this did not reach statistical significance.!0, 百拇医药
fig.ommitted!0, 百拇医药
Figure 6. Effect of hEC-SOD overexpression on lung myeloperoxidase accumulation in hyperoxia-exposed newborn mouse at 21 days. n = 5 per group. Mean + SEM, *p value less than 0.05 versus transgenic, hyperoxia-exposed.!0, 百拇医药
EC-SOD activity.!0, 百拇医药
EC-SOD transgenic mice maintained elevated total lung EC-SOD activity at 21 days. Transgenic animals had more than threefold the activity levels of those observed in wild-type littermates, as in the 7-day exposed newborns . Hyperoxia-exposure induced an additional increase in total EC-SOD activity compared with air-exposed transgenic animals. Hyperoxia had no effect on EC-SOD activity in wild-type littermates.
fig.ommittedt|, http://www.100md.com
Figure 7. EC-SOD activity in mouse lung at 21 days, air versus chronic hyperoxia, wild-type versus transgenic. Mean + SD of three replicate measurements from pooled samples, n = 4 per group, *p value less than 0.05 versus wild-type, p value less than 0.05 versus EC-SOD air.t|, http://www.100md.com
DISCUSSIONt|, http://www.100md.com
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REFERENCESt|, http://www.100md.com
Overexpression of human EC-SOD in hyperoxia-exposed adult mice confers improved survival and decreased inflammation (17). In our model of chronic lung disease, we sought to use a sublethal injury that shares some features of human chronic lung disease, namely oxidant stress, inflammation, and impaired alveolar development (16). We found that overexpression of human EC-SOD targeted to pulmonary epithelium partly prevented glutathione oxidation, and reduced hyperoxia-induced interstitial and airway inflammation at 7 days. Later alveolar development—as determined by alveolar surface and volume density—was preserved in hEC-SOD transgenic mice exposed to hyperoxia for 21 days.
EC-SOD expression may confer particular protection against oxidant stress. Mutant mice lacking EC-SOD are more susceptible to hyperoxia-induced injury and inflammation (18). Transgenic mice overexpressing mitochondrial (manganese) SOD showed improved survival and attenuation of acute injury (16). Hyperoxia-exposure studies in transgenic mice overexpressing cytosolic (copper–zinc) SOD conferred similar benefits (27). Because EC-SOD is a secreted protein, it may dismutate superoxide in vulnerable lung compartments relatively inaccessible to intracellular antioxidants, such as the surface of alveolar epithelium or the alveolar hypophase (28).r., 百拇医药
Inflammation may exacerbate oxidant stress and thus contribute to propagation of lung injury, and it may impede repair and cell proliferation. Our previous studies in hyperoxia-exposed adult EC-SOD overexpressing mice showed attenuation of inflammatory cytokines and adhesion molecules (17). We have recently shown that blockade of neutrophil influx protects against adverse hyperoxia effects on lung development in newborn rats (26). The preservation of alveolar development we observed at 21 days in the present studies may be partly attributable to the early reduction of inflammation we observed as reduced BAL fluid leukocyte counts and tissue myeloperoxidase in transgenic mice at 7 days.
Overexpression of EC-SOD preserved lung development during oxidative stress, evident as partly preserved alveolar surface and volume density. This would indicate that secondary septation is likely preserved because decreased septation would result in lower surface density. There were no differences in surface density between air-exposed newborn wild-type and transgenic mice. This suggests that EC-SOD transgenic animals do not have constitutively superior alveolar development in the absence of oxidative stress.2qw, 百拇医药
The beneficial effects of EC-SOD overexpression may be mediated indirectly by alterations in ROS signaling of cellular processes. Hydrogen peroxide, produced spontaneously or by SOD by single electron reduction of superoxide, has been increasingly recognized as a mediator of in vitro apoptosis and may alter cell proliferation (29). Reduction of hydrogen peroxide by exogenous catalase in vitro can abrogate the effect of cellular mitogens, as recently reviewed by Jankov and coworkers(30). Potential effects of SOD on hydrogen peroxide–mediated signaling would likely depend greatly on the timing, concentration, and location of hydrogen peroxide.
Hyperoxia induced even higher levels of total EC-SOD activity in the transgenic animals at 7 and 21 days. The transferred hEC-SOD gene is under control of the human SP-C promoter in the transgenic mice. It is known that hyperoxia induces increased SP-C messenger RNA in mice, and we speculate that similar mechanisms may be enhancing the human SP-C promoter–controlled transgenic EC-SOD overexpression (31, 32). In contrast, human EC-SOD protein levels measured by immunoblot did not appear to be induced by hyperoxia, but this was not strictly quantified. Cellular protein processing in the transgenic mice may alter the EC-SOD activity without changing the total lung EC-SOD accumulation. The wild-type littermates showed no hyperoxia-induced changes in either EC-SOD activity or mouse EC-SOD accumulation in whole lung.g, http://www.100md.com
We conclude that overexpression of hEC-SOD in neonatal mice protects against hyperoxia-induced inflammation at 7 days and impairment of lung development at 21 days. Although the specific mechanism is still unclear, it likely includes preservation of reduced glutathione—important for lung oxidant–antioxidant equilibrium—and attenuation of inflammation that may cause cell death and alveolar destruction. Newborn mice exposed to hyperoxia for 21 days showed maintenance of both alveolar volume and surface density. We speculate that correctly targeted SOD overexpression will prevent adverse effects of oxidant stress on lung development and may protect lung development in newborns at risk to develop BPD.
Acknowledgments+'3, 百拇医药
The authors gratefully acknowledge the technical assistance of Mary Whorton and Kathryn Auten.+'3, 百拇医药
REFERENCES+'3, 百拇医药
TOP+'3, 百拇医药
ABSTRACT+'3, 百拇医药
METHODS+'3, 百拇医药
RESULTS+'3, 百拇医药
DISCUSSION+'3, 百拇医药
REFERENCES+'3, 百拇医药
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