Introduction

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Bronchopulmonary dysplasia (BPD) is a chronic lung disease that develops in newborn infants treated with oxygen and mechanical ventilation for a primary lung disorder (1). BPD affects approximately 20 to 60% of premature infants and is strongly associated with significant morbidity and mortality. BPD has become an extremely important complication of neonatal intensive care due to the increasing survival of infants with very low birthweight (2). The etiology of BPD is multifactorial; however, hyperoxia and barotrauma from positive pressure mechanical ventilation are believed to be important factors involved in the pathogenesis. Hyperoxia is known to be associated with increased production of reactive oxygen species (ROS) that can damage the structurally immature lung. In addition, premature infants are relatively deficient in both endogenous surfactant and protective antioxidant enzymes at birth, which may further contribute to the lung injury process (3).

Preliminary animal and human studies have suggested that acute and chronic lung injury from hyperoxia and mechanical ventilation may be ameliorated by the administration of one of these antioxidants, specifically superoxide dismutase (SOD) (4). There are three different forms of SOD that have been found in mammalian cells: cytosolic copper zinc SOD (CuZnSOD), a mitochondrial manganese SOD (MnSOD), and an extracellular CuZnSOD. The only known function of the enzyme is to catalyze the conversion of toxic superoxide anions to potentially less toxic hydrogen peroxide and water. Catalase (CAT) is an enzyme responsible for converting hydrogen peroxide to oxygen and water. Interestingly, SOD and CAT activities have been detected in natural lung surfactant, but they are absent in commercial preparations (7). Clinical trials in premature infants receiving exogenous surfactant, mechanical ventilation, and oxygen therapy for respiratory distress syndrome have demonstrated significant reductions in acute inflammatory changes and longer-term improvements in clinical pulmonary status in infants also receiving multiple intratracheal doses of recombinant human (rh) CuZnSOD (8, 9). It is unclear whether supplementation with other antioxidant enzymes such as CAT in addition to rhCuZnSOD would further protect the lung against oxidant injury.

Because the lung is susceptible to injury from hyperoxia, MLE 12 cells were used in the present study as a model of type II pneumocytes. These are transformed cells that have several morphologic and biochemical features of type II cells, including polygonal epithelial cell morphology, microvilli, and cytoplasmic multivesicular bodies. In addition, these cells have been shown to express surfactant protein (SP)-B and SP-C messenger RNAs (mRNAs) (10). To determine if the combination of MnSOD and CAT could provide more optimal protection to pulmonary epithelial cells against oxidant injury compared with either antioxidant alone, we generated stable MLE 12 cell lines, overexpressing these genes either alone or in combination. The viability of the various cell lines was determined after exposure to prolonged hyperoxia or other oxidant challenges such as paraquat.

	Materials and Methods

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Construction of MnSOD and CAT Complementary DNA Plasmids

MnSOD complementary DNA (cDNA) was prepared from HeLa-20 cells by reverse transcriptase-polymerase chain reaction (RT-PCR). PCR amplification of human MnSOD cDNA was performed using a sense primer flanked by an EcoRI restriction site (5'-GAATTCAGCAGCATGTTGAGCCGCG-3') and an antisense primer flanked by a NotI restriction site (5'-GCGGCCGCTTACTTTTTGCAAGCCATGTATC-3'). The entire protein coding region of MnSOD cDNA (bases +95 through +763) was amplified (GeneBank accession no. Y00985). This region included the signal peptide (bases +95 through +166). PCR amplification of human CAT cDNA (no. 403830; American Type Culture Collection, Rockville, MD) was performed with primers 5'-GGGGTACCAAGCTTCACGCTATGGCTGACAGCCGG-3' and 5'-TCTAGAGCGGCCGCTCACAGATTTGCCTTCTCCCTTG-3'. PCR products were gel purified and MnSOD and CAT cDNAs were then cloned into pGEM-T System I (Promega, Madison, WI). Fragments encoding the entire coding region of the genes were excised for cloning into the vectors used for selection. MnSOD cDNA was digested with EcoRI and NotI, and CAT cDNA was digested with KpnI and XbaI. Inserts were isolated and ligated into one of two vectors: pOPRSVI/MCS (Stratagene, La Jolla, CA) or pWE-4 (CAT). Insertion in pOPRSVI/MCS was downstream of the Rous sarcoma virus promoter plus intron and upstream of the thymidine kinase polyA signal. Insertion in the pWE vectors was downstream of the cytomegalovirus promotor and upstream of the simian virus 40-early polyA signal and small T-intron (11).

Generation and Screening of Stable Cell Lines

MLE 12 cells were grown in Hites medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, and maintained at 95% room air, 5% CO₂ in a humidified chamber. Cells were transfected with Lipofectamine plus Reagent (Life Technologies, Grand Island, NY). Stable cell lines were generated containing vector with no insert, CAT, MnSOD, and MnSOD + CAT. Stable cell lines were initially screened based on resistance to the appropriate antibiotic (200 µg/ml of gentamycin for pOPRSV1 and 100 µg/ml of hygromycin for pWE-4). At least 10 colonies per group were isolated and screened for enzymatic activity. Three overexpressing cell lines from each group were randomly selected for further study. The antioxidant enzyme activities were stable in these cell lines for 10 to 15 passages.

Enzymatic Assays

Enzymatic activities of MnSOD and CAT were measured spectrophotometrically as previously described (12, 13). A unit of SOD activity was defined as the amount of SOD required to inhibit the reduction of cytochrome c at 25°C and pH 7.8 by 50%. One unit of CAT activity was defined as the amount of CAT required to decompose 1 µmol of hydrogen peroxide in 1 min at 25°C and pH 7.0.

Exposure to Hyperoxia and Superoxide: Viability and Clonogenic Assays

Cells were seeded at 35% confluence and allowed to adhere overnight. The cells were then exposed to 95% O₂, 5% CO₂ for up to 10 d or varied concentrations of paraquat (Sigma Chemical Co., St. Louis, MO) for 24 h. Media and gases were refreshed daily when cells were cultured for several days. Cell viability was determined by the exclusion of trypan blue dye and counted using a hemacytometer. All experiments were performed in triplicate. Clonogenic capability was assessed after cells were exposed to hyperoxia for designated amounts of time (1, 3, or 5 d). The cells were then washed with phosphate-buffered saline (PBS), trypsinized, and reseeded on 100-mm plates at 200 cells per plate. Cells were incubated in room air and 5% CO₂ at 37°C for up to 5 d. The plates were then washed with PBS and cells fixed with cold methanol for 3 min and stained with methylene blue. Colonies were counted under a dissecting microscope.

Statistical Analyses

Differences in survival and enzymatic activities between the cell lines were compared using two-way analysis of variance with Bonferroni correction for multiple pairwise comparisons. Correlation between cell survival and enzymatic activities was analyzed with Pearson and Spearman correlation coefficients. A P value of less than 0.05 was accepted as statistically significant. All analyses were performed using the SAS software package version 6.12 (SAS Institute, Cary, NC).

	Results

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To assess the amount of the overexpression from the transgene, we used enzymatic activity assays and the comparison to parental, untransfected cells as shown in Table 1. Cells overexpressing MnSOD alone showed a 65 to 98% increase (range of all three clones) in MnSOD activity (P < 0.05). Cell lines overexpressing CAT alone showed a 40 to 52% increase in CAT activity (P < 0.05). Cells overexpressing both MnSOD and CAT showed a 66 to 173% increase in MnSOD activity and a 33 to 64% increase in CAT activity (P < 0.05).

To test whether overexpression of MnSOD protects lung epithelial cells against hyperoxia, cells were exposed to 95% oxygen for up to 10 d. Figure 1A shows that after 5 d of hyperoxia, all cell lines overexpressing MnSOD (MLMn-0, -4, and -5, and MLMnCAT-0, -7, and -9) had significantly improved survival compared with parental and vector-only control cells (MLE 12 and MLV-0) (P < 0.001). Pearson correlation analysis revealed a strong correlation (r = 0.86) between overexpression of MnSOD and cell viability (Figure 2A). In contrast, overexpression of only CAT offered no additional protection from oxidant injury (Figures 1 and 2B). Overexpression of both MnSOD and CAT did significantly improve survival compared with MnSOD alone (P < 0.001). Similar results were obtained after 10 d of exposure to 95% O₂ (Figure 1B). MLE 12 and MLV-0 cells were growth arrested in hyperoxia in a similar fashion to other transformed type II cell lines (14, 15). Although cells overexpressing CAT were also growth arrested, those overexpressing MnSOD (with/without CAT) continued to divide over 24 to 48 h (never reaching confluence).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Comparison of cell viability in 95% O2. Cells were exposed to 95% O2 for 5 d (A) or 10 d (B) and viability was assayed by dye exclusion. All samples were performed in triplicate. Data represent mean ± SEM. *Statistically different from control (MLE 12) cells (P < 0.001). **Statistically different from MnSOD-only cells (P < 0.001).

View larger version (14K): [in this window] [in a new window]   Figure 2.   Correlation analysis of MnSOD and CAT activity with resistance to hyperoxia. (A) Scattergram plot of MnSOD activity (percent of parental control cells) versus cell viability correlating resistance to hyperoxic injury with MnSOD activity after 5 d of exposure to hyperoxia. (B) Scattergram plot of CAT activity (percent of parental control cells) versus cell viability demonstrating no resistance with increased CAT activity after 5 d of exposure to hyperoxia.

To assess the growth potential of the surviving cells, a clonogenic assay was performed on representative cell lines. Cells overexpressing MnSOD (MLMn-0 and MLMnCAT-0) demonstrated significantly improved growth potential compared with vector control cells, and cells overexpressing only CAT (P < 0.05) at all the exposures (1, 3, and 5 d) in hyperoxia (Figure 3). In this instance, the overexpression of both CAT and MnSOD did not significantly improve the clonogenic potential compared with overexpression of MnSOD alone.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Growth potential of cells after exposure to 95% O2. Cells from representative cell lines were exposed to 95% O2 for 1 d (solid bars), 3 d (shaded bars), and 5 d (open bars) then assessed for their growth potential. The experiment was performed in duplicate and the data represent the mean ± standard deviation. *Statistically different from vector controls (P < 0.05).

To examine the cellular response to other types of oxidants, we exposed cells to paraquat (a known intracellular superoxide generator). In contrast to hyperoxia, paraquat kills cells more rapidly over the course of hours rather than days. Cells were exposed to 1 or 5 mM of paraquat for 24 h and the results are illustrated in Figure 4. Overexpression of MnSOD increased survival at either concentration of paraquat, whereas CAT overexpression offered no protection (Figure 5). Even at relatively high doses (5 mM), cells overexpressing MnSOD (± CAT) had a two- to threefold increase in survival compared with the parental (MLE 12) and vector-only cells (Figure 4B). In contrast to hyperoxia, overexpression of both MnSOD and CAT did not improve survival over MnSOD alone (Figures 4 and 5). Analysis of the results revealed a correlation between MnSOD activity and cell survival in these experiments (Figure 5A; P < 0.001) but no correlation with CAT overexpression (Figure 5B).

View larger version (21K): [in this window] [in a new window]   Figure 4.   Comparison of cell viability of cells in paraquat. Cells were exposed to 1 (A) or 5 mM (B) paraquat for 24 h and viability was assayed by dye exclusion. All samples were performed in triplicate. Data represent mean ± SEM. *Statistically different from control (MLE 12) cells (P < 0.001).

View larger version (16K): [in this window] [in a new window]   Figure 5.   Correlation analysis of MnSOD and CAT activity with resistance to 1 mM paraquat. (A) Scattergram plot of MnSOD activity (percent of control cell lines) versus cell viability correlating resistance to paraquat with MnSOD activity. (B) Scattergram plot of CAT activity (percent of control cell lines) versus cell viability demonstrating no resistance with CAT activity.

	Discussion

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In the present study, we demonstrate that overexpression of MnSOD provides significant protection to alveolar epithelial cells from oxidant injury, whereas CAT alone does not. Furthermore, overexpression of both MnSOD and CAT does provide additional benefits against hyperoxic injury compared with MnSOD alone.

Several laboratories have studied the regulation of antioxidant enzymes in various cell culture model systems. Overexpression of MnSOD in bovine lung microvascular endothelial cells exposed to hyperoxia for 24 h resulted in a twofold increase in cell survival compared with control cells (16). The overexpression of multiple antioxidant enzymes has been previously studied by Tiedge and coworkers (17). Their results demonstrate that overexpression of CAT and glutathione peroxidase, alone or in combination with CuZnSOD in the insulin-producing cell line RINm5F, can protect cells against oxidative damage induced by hydrogen peroxide, hypoxanthine/xanthine oxidase, and menadione. Interestingly, overexpression of CuZnSOD alone provided no additional protection in their studies (17). SOD also has been shown to have a protective role in cytokine-induced cell death. For instance, a 24-h exposure of mouse endothelial cells to tumor necrosis factor- demonstrated increases in MnSOD mRNA with a corresponding increase in enzyme activity (18). In addition, stable overexpression of MnSOD in pancreatic islet  cells resulted in complete protection against interleukin-1-mediated cytotoxicity (19).

Important insights on the protective role of antioxidant enzymes during hyperoxia have also been gained through animal and human studies. For example, transgenic mice with disrupted extracellular CuZnSOD genes have markedly increased mortality and lung damage when exposed to prolonged hyperoxia (20). Conversely, genetically engineered mice that overexpress MnSOD only in type II pneumocytes were able to survive longer and have decreased mortality and pulmonary damage when exposed to hyperoxia (21). A number of animal studies have shown significant improvements in lung morphology and survival from prolonged hyperoxia through the use of intravenous, intraperitoneal, or intratracheal administration of CuZnSOD (22). In addition, clinical trials have demonstrated decreased inflammatory changes and long-term pulmonary injury in premature infants with respiratory distress syndrome that received rhCuZnSOD (8). These findings suggest that increased production or supplementation with antioxidant enzymes in the lung mitigates lung damage from ROS. The results of the present study clearly demonstrate that MnSOD overexpression provides protection from both hyperoxia and superoxide-induced injuries. However, data also indicate that supplementation of CAT in addition to SOD may enhance pulmonary protection to ROS-mediated injury.

Our interest in studying MnSOD was supported by evidence that the mitochondria are the major site of oxygen radical production in the cell that increases in direct proportion to oxygen tension (25). Electron microscopic studies have demonstrated that the mitochondrion is an early target of oxygen injury, undergoing swelling, loss of cristae, and eventually disruption of mitochondrial membranes in the presence of hyperoxia. Oxygen injury attenuates adenosine triphosphate (ATP) production, which may impair the ability of the cell to repair oxidant damage to other cellular components (26). Mitochondria play a key role in regulating cell death caused by both apoptosis and necrosis. Changes in membrane permeability transition and cytochrome C release from the mitochondria initiate signal transduction pathways that regulate apoptosis, whereas necrosis is thought to occur when a cell depletes its ATP stores. Because MnSOD is located in the mitochondrial matrix, it would appear to be strategically advantageous to provide an initial defense against locally produced superoxide radical. It is unclear whether the location of the enzyme versus overall activity is important for the protection of the cell.

The cells used in this study were derived by the isolation of tumors generated in transgenic mice designed to isolate respiratory epithelial cell lines (27). Although there are differences between nontransformed versus transformed epithelial cells, given the complexity of the lung, using transformed cell lines is a powerful tool for delineating which combination of antioxidant enzymes offers the best protection and the mechanism of protection from oxidant injury. Given that the overexpression protection of MnSOD in airway epithelium mitigates injury from hyperoxia in vivo, it is highly unlikely that the protection reported here is an anomaly of transformation.

SOD converts highly toxic superoxide to potentially less toxic hydrogen peroxide. However, in the presence of ferric iron, hydroxyl radicals (highly reactive ROS) can be generated that can cause significant tissue injury (28). Therefore, it would appear to be critical to have sufficient CAT present to facilitate the conversion of H₂O₂ to oxygen and water. In fact, although overexpression of MnSOD improved cell survival from oxidant injury, the addition of CAT to cell lines overexpressing MnSOD further enhanced cell survival from prolonged hyperoxia. Because these were in vitro studies performed under controlled conditions, it is possible that adequate CAT is present in vivo (red blood cells, etc.) to detoxify the additional H₂O₂ generated when supplemental SOD is administered to premature infants.

Our long-term goal is to develop new therapies for the prevention and management of BPD. Although the pathogenesis of BPD is complex and multifactorial, it is currently thought that pulmonary oxygen toxicity plays a prominent role (29). Premature infants often require treatment with supraphysiologic concentrations of oxygen for respiratory distress syndrome, but hyperoxia is toxic, especially to lungs. The toxicity of hyperoxia suggests that endogenous levels of antioxidants are insufficient to cope with this insult. Under hyperoxic conditions there is an increased production of reactive oxygen metabolites, including superoxide radical (O₂), hydrogen peroxide (H₂O₂), and hydroxyl radical (·OH). These oxygen species disrupt biomembranes, inhibit cellular enzymes, and shift cellular redox state toward oxidation and impaired energy production. Protection against the cascade of oxidizing metabolites requires a system of substrate-specific antioxidant enzymes. Networks of multilayered, mutually supporting enzymatic and nonenzymatic antioxidants have evolved to detoxify highly reactive oxygen-derived radicals that are natural byproducts of aerobic metabolism. It is apparent that an imbalance between oxidants and antioxidants is involved in the pathogenesis of BPD. Delineating the optimal combination of antioxidant enzymes that will best protect epithelial cells in the lung from oxidant injury may lead to novel therapies for the prevention of BPD in premature infants and for other patients requiring treatment with increased oxygen.