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1Vascular Biology Center, Medical College of Georgia, Augusta, Georgia; 2Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana; and Departments of 3Pediatrics, 4Surgery, and the 5Cardiovascular Research Institute, University of California, San Francisco, California
Submitted 14 November 2006 ; accepted in final form 26 June 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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catalase; superoxide dismutase; congenital heart disease
Antioxidant enzyme systems play a critical role in the regulation of oxidant levels in the vasculature, and their disregulation has been implicated in the pathobiology of systemic hypertension (26). There are several antioxidant systems regulating the presence of oxidant species in vivo, but of primary interest are the superoxide dismutases (SOD) and catalase. There are two SOD in cells, Cu/ZnSOD (SOD1) in the cytoplasm and MnSOD (SOD2) in the mitochondria, that serve to catalyze the rapid conversion of superoxide into H2O2 and oxygen, whereas catalase breaks down H2O2 into oxygen and water (26). In addition, there is a third SOD isoform located on the extracellular surface, EC-SOD (SOD3). Together, these systems serve to keep oxidant levels low thereby reducing the overall oxidant stress within the cells. In experimental models of systemic hypertension, the administration of antioxidants has been shown to ameliorate endothelial dysfunction and attenuate the disease state (15, 17). In addition, reduced antioxidant activity/levels have been demonstrated to contribute to oxidative stress in patients with systemic hypertension (25).
Although the effects of oxidative stress in the development of systemic hypertension have been characterized, less is known regarding the developmental expression of antioxidant enzymes in normal pulmonary postnatal development and in pulmonary hypertensive disorders (2, 3). In addition, studies on the pulmonary vasculature have focused on advanced pulmonary hypertension, while potential changes in ROS during the early development of pulmonary hypertension have not been investigated (5). Therefore, the objective of this study was to characterize the expression patterns of antioxidant enzymes in normal postnatal pulmonary development, and during the early development of pulmonary hypertension secondary to increased pulmonary blood flow. To determine expression patterns of antioxidant enzymes in normal postnatal development, lung tissue protein and activity levels of catalase, SOD1, and SOD2 were determined in 2-, 4-, and 8-wk-old lambs. To determine expression patterns of antioxidant enzymes during the early development of pulmonary hypertension secondary to increased pulmonary blood flow, lung tissue protein and activity levels of catalase, SOD1, SOD2, and SOD3 were determined in 2-, 4-, and 8-wk-old lambs with increased pulmonary blood flow, secondary to in utero placement of an aortopulmonary vascular graft.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2-mm length; W. L. Gore and Assos., Milpitas, CA) was anastomosed between the ascending aorta and main pulmonary artery with 7.0 prolene (Ethicon, Somerville, NJ) and the thoracotomy incision was then closed in layers. The incisions in the uterus and the abdomen were closed, and the sheep were allowed to deliver normally. This procedure is previously described in detail (20).
Two, four, and eight weeks after spontaneous delivery, lambs were anesthetized with ketamine hydrochloride (0.3 mg·kg–1·min–1), diazepam (0.002 mg·kg–1·min–1), and fentanyl citrate (1.0 µg·kg–1·h–1), intubated, mechanically ventilated, and a midsternotomy incision was performed. Four biopsies of peripheral lung tissue were harvested from randomly selected lobes, and 300 mg of peripheral lung were obtained for each biopsy. Oxygen saturations were obtained in the aorta, right ventricle, right atrium, and distal pulmonary artery to confirm graft patency and increased pulmonary blood flow.
At the end of the protocol, all lambs were killed with a lethal injection of pentobarbital sodium followed by bilateral thoracotomy as described in National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco.
Western blotting. Lung tissue was homogenized in Triton X-100 lysis buffer [20 mM Tris·HCl (pH 7.6), 0.5% Triton X-100, 20% glycerol] supplemented with protease inhibitors (100 µg/ml PMSF, 1 µg/ml leupeptin and aprotinin), clarified by centrifugation at 20,000 g for 20 min at 4°C, and the supernatant was stored at –80°C until needed. Before use, the protein concentration was quantified by the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA).
Twenty-five to fifty micrograms of total protein per lane were loaded onto Long-Life 4–20% Tris-SDS-HEPES gels (Gradipore, Frenchs Forest, Australia) and run to completion according to the manufacturer's instructions. The proteins were transferred to Immun-Blot PVDF membrane (Bio-Rad Laboratories) and the membrane was blocked with 5% skim milk in TBS-T for 1 h to overnight. The membranes were probed with antibodies to SOD1 (1:1,000), SOD2 (1:1,000, Upstate, Lake Placid, NY), SOD3 (1:250, StressGen, Ann Arbor, MI), and catalase (Research Diagnostics, Flanders, NJ). Reactive bands were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate Kit after incubation with an anti-rabbit-horseradish peroxidase secondary (1:1,000, Pierce, Rockford, IL) and Kodak 440CF image station (Kodak, New Haven, CT). The intensity of the reactive bands was quantified using the Kodak 1D software. Expression of each protein was normalized by reprobing each gel with 
-actin (Sigma, St. Louis, MO).
Confocal immunohistochemistry. Snap-frozen lung tissue samples were embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA), cryosectioned at 5 µm, collected onto Superfrost Plus slides (VWR Scientific, West Chester, PA), allowed to air-dry at room temperature, and stored at –80°C until needed. Double-labeling immunofluorescence was performed on serial sections of control and shunt ovine lung using SOD2 (1:100), SOD3 (1:50), and catalase (1:100). Localization to the endothelium or smooth muscle layer was confirmed by double labeling each section with either mouse anti-endothelial NO synthase (eNOS; 1:100, BD Transductions Labs, Lexington, KY) or mouse anti-caldesmon (1:500, Sigma). Fresh-frozen tissue sections were allowed to come to room temperature, washed in phosphate buffer saline (PBS), and fixed in ice-cold acetone for 10 min. Sections were air-dried for 1 h, permeabilized in PBS with 0.1% Triton X-100 for 10 min, blocked in 10% normal goat serum overnight at 4°C, and then incubated in primary antibody for 1 h at room temperature. Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse antibodies (1:250 dilution for each, Molecular Probes) were used for detection of inducible (i)NOS and eNOS/smooth muscle caldesmon, respectively. Sections were washed several times in PBS, mounted, and coverslipped in anti-fading aqueous mounting medium.
The confocal images were taken on a Zeiss LSM 510 Meta confocal laser-scanning microscope using Plan-Neofluar 40x/1.3 oil DIC objective and Argon 488 and HeNe 543 lasers. A series of images at 1-µm interval focal planes were collected into a single file or z-stack to determine actual colocalization. Recorded images were processed using Adobe Photoshop software.
SOD activity assay. Frozen (–80°C) lung tissue was weighed and 5x the weight of 10 mM EDTA added, and the tissue was minced and sonicated with 3 x 10-s pulses at 40% power (High Intensity Ultrasonic Processor, Autotune series, A. Daigger and Co, Vernon Hills, IL). The samples were centrifuged at 20,000 g, 4°C for 15 min, the supernatant was collected, and aliquots were frozen at –80°C until needed. The protein content of the homogenates was determined by the Bio-Rad DC Protein Assay (Bio-Rad Laboratories).
Tissue SOD activity was determined by a modification of the method of Elstner and Heupel (4) using bovine liver SOD (Sigma) to generate the standards. Performing the assay in the absence and presence of KCN allows for the determination of the total SOD and SOD2 activity, respectively, while the SOD1 activity is the difference between the activities. Briefly, sample tubes and SOD standards contained potassium phosphate dibasic solution (pH 7.8), dH2O, xanthine, hydroxylamine HCl, SOD standard from bovine liver or sample and freshly prepared xanthine oxidase (for total SOD activity). Duplicate tubes contained 10 mM KCN in the potassium phosphate solution in addition to the remainder of the components (for Mn SOD activity). The samples were incubated at room temperature for 20 min and then placed on ice to stop the reaction. Sulfanilic acid and 1-Napthlamine were added to all wells, mixed well, and reacted at room temperature for 20 min in the dark. This assay gives a red reaction product where less red color equates to more SOD activity and was read at 550 nm.
Catalase assay.
Catalase activity was quantified in lung tissue (prepared as per Western blotting above) by a modification of the method of Aebi (1). For each sample, 40 µg of protein were added to 50 mM potassium phosphate buffer, pH 7.0, and 3% H2O2 (10 mM) was added and the consumption was followed at 240 nm for 60 s using the Shimadzu UV-1700 Pharmaspec and UV Probe software (Shimadzu, Columbia, MD). The change in absorbance was used to calculate catalase-specific activity using the extinction coefficient of 43.6 mM/cm for H2O2.
In situ detection of H2O2. Snap-frozen lung tissue samples stored at –80°C were embedded in Tissue-Tek O.C.T Compound (Sakura Finetek USA), cryosectioned at 20 µm, and collected onto Superfrost plus slides (VWR Scientific), allowed to air-dry at room temperature, and stored at –80°C until needed. For staining, slides were removed from –80°C and placed into PBS for 30 min at room temperature. H2O2 levels in lung tissue slices were determined by the change in fluorescence resulting from the oxidation of the fluorescent probe, dichlorodihydrofluorescein diacetate (H2DCF-DA; Calbiochem) in serial sections of control and shunt ovine lung. Frozen tissue sections were allowed to come to room temperature and H2DCF-DA (10 µM) for 30 min at room temperature in the dark. PEG-catalase (100 units) was used to confirm that the oxidation of H2DCF-DA was H2O2 dependent. The slides were rinsed extensively with PBS, coverslipped, and imaged with an Olympus IX51 inverted microscope. In all cases, exposure time was kept constant between slides. Values were averaged from multiple vessels captured within each field and care was taken to identify vessels within nonadjacent areas to prevent photobleaching. Average fluorescent intensities (to correct for variability in slice thickness) were quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Data are presented as PEG-catalase-inhibitable H2DCF-DA fluorescence.
Electronic paramagnetic resonance measurement of superoxide anions in lung tissue. Approximately 0.2 g of tissue was sectioned from fresh-frozen samples of fourth generation PA and immediately immersed in either normal electronic paramagnetic resonance (EPR) buffer [PBS supplemented with 5 µM diethydithiocarbamate (DETC); Sigma] and 25 µM desferrioxamine (Def MOS, Sigma). Samples were incubated for 30 min on ice. During incubation, samples were analyzed for protein content using Bradford analysis (Bio-Rad). Sample volumes were then adjusted with EPR buffer + 25 mg/ml 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl (CMH hydrochloride, Axxora) to achieve equal protein content and a final CMH concentration of 5 mg/ml. Samples were homogenized for 30 s with a VWR PowerMAX AHS 200 tissue homogenizer, incubated for 60 min on ice, and then centrifuged at 14,000 g for 15 min at room temperature. Thirty-five microliters of supernatant were loaded into a 50-µl capillary tube and analyzed with a MiniScope MS200 ESR (Magnettech, Berlin, Germany) at a microwave power of 40 mW, modulation amplitude of 3,000 mG, and modulation frequency of 100 kHz. EPR spectra were analyzed, measured for amplitude using ANALYSIS v.2.02 software (Magnettech), and experimental groups were compared using statistical analysis.
Statistical analysis. Statistical calculations were performed using the GraphPad Prism V. 4.01 for Windows, GraphPad Software (San Diego, CA; www.graphpad.com). The means ± SD or SE were calculated for all samples and significance was determined by either the unpaired t-test (for 2 groups) or ANOVA with Bonferroni's post hoc test (for 3 or more groups). A value of P < 0.05 was considered significant.
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RESULTS |
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Catalase protein levels. The expression of catalase protein was evaluated by Western blotting to determine whether there were differences between control and shunt lambs. Catalase expression was decreased in the 2-wk shunts compared with 2-wk controls (Fig. 1, A and B), while no differences were seen between controls and shunts at 4 (Fig. 1, A and B) and 8 wk (Fig. 1, A and B). Postnatally in control lambs, catalase expression was increased by about threefold at 4 wk, compared with 2- and 8-wk animals (Fig. 1, C and D). In shunt lambs, catalase protein levels were increased by about threefold in 4- and 8-wk lambs compared with 2-wk lambs (Fig. 1, C and D).
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Catalase localization within pulmonary arteries. To determine whether there were differences in catalase localization within either the endothelial or smooth muscle layers of pulmonary arteries in 2-wk-old shunt and control lambs, we utilized dual-labeleing fluorescent immunohistochemistry to determine catalase expression in either the endothelium (colocalization with eNOS) or the smooth muscle (colocalization with caldesmon). The data obtained indicate that catalase is highly expressed within both the endothelial (Fig. 2A) and smooth muscle layers (Fig. 2B) and we could not detect any preferential differences in expression within the shunt lambs that would explain the decrease in catalase expression in shunt lambs observed by Western blot analysis (Fig. 1, A and B).
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7- and 7.6-fold, respectively (Fig. 4, C and D).
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SOD2 protein levels. SOD2 expression was decreased in 2-wk shunt compared with control lambs (Fig. 5, A and B), while no significant differences were seen in SOD2 expression between controls and shunt lambs at 4 and 8 wk of age (Fig. 5, C and D). Postnatally in control lambs, SOD2 expression was increased at 4 and 8 wk compared with 2 wk of age (Fig. 5, C and D). Postnatally in the shunt lambs, SOD2 was increased at 8 wk compared with 2 wk of age (Fig. 5, C and D).
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SOD2 activity. SOD2 activity was increased developmentally in control lambs, such that activity was increased at 4 and 8 wk compared with 2 wk of age, but was not developmentally altered in shunt lambs (Fig. 5G). However, compared with control lambs, SOD2 activity was higher at 4 wk of age in shunt lambs (Fig. 5G).
SOD3 protein levels. SOD3 expression was increased in 2-wk shunt compared with control lambs (Fig. 6, A and B), while no significant differences were seen in SOD3 expression between controls and shunt lambs at 4 and 8 wk of age (Fig. 6, A and B). When developmental expression of SOD3 was quantified in the control and shunt lambs, no differences were seen in the control or shunt lambs at any time points (Fig. 6, C and D).
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Lung superoxide levels. Superoxide quantification, as determined by EPR on peripheral lung from shunt and control lambs at 2, 4, and 8 wk of age, is shown in Fig. 7. Superoxide levels were found to be significantly higher in shunt compared with control lambs at 2 and 4 wk (Fig. 7A). However, this differnce was not sustained at 8 wk of age (Fig. 7A). In addition, superoxide levels were found to increase from 2 to 8 wk of age in both shunt and control lambs (Fig. 7, B and C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Several enzyme systems are present in organisms that serve to detoxify the ROS and prevent or reduce oxidative damage. The major antioxidant enzyme systems responsible are SOD and catalase, which remove superoxide and H2O2, respectively. In the present study, during the first 2 mo of postnatal development, we found a transient increase in catalase protein expression at 4 wk of age, which was not associated with changes in catalase activity. The fact that catalase expression does not correlate with catalase activity suggests that there may be posttranslational regulation of the protein. One potential explanation is that there may be peroxynitrite-mediated inhibition of catalase at 4 wk of age. We previously showed that there is an increase in NOS-dependent ROS in 4-wk-old shunt lambs (8), whereas a previous study indicated that purified catalase is inhibited when exposed to peroxynitrite (14). However, further studies will be required to determine whether there is an increase in peroxynitrite-mediated catalase nitration in the 4-wk-old shunt lambs.
There are very limited studies that have investigated developmental pulmonary catalase expression. However, in the human lung, catalase mRNA has been shown to increase from the fetus into adulthood (3). Interestingly, under conditions of increased pulmonary blood flow, catalase expression and activity are initially decreased, but they increase during the second month of life to equal those of normal age-matched controls and this decrease correlates with an increase in lung H2O2 levels as estimated using H2DCF-DA fluorescent imaging. However, at subsequent ages (4 and 8 wk) catalase expression in shunt lambs was not different from age-matched controls. These data extend to our previous studies in which at 4 wk of increased pulmonary blood flow, we showed that superoxide levels are increased in shunt lambs, but H2O2 levels are unchanged (8). The current data suggest that the increase in catalase expression in shunt lambs after 2 wk of age may limit the accumulation of H2O2 in this setting. However, the mechanisms that regulate catalase expression under these conditions are unclear and require further study.
Prior studies in the developing human lung (17-wk gestation) have indicated that catalase expression is limited to the bronchial epithelium and to alveolar macrophages (13). However, our immunohistochemical data indicate that in the postnatal lamb lung, there is robust expression of catalase in both the endothelium and the smooth muscle layers of pulmonary arteries. Although it is difficult to make comparisons between species given the potential differences in developmental maturation, this suggests that there may be a postnatal increase in catalase within pulmonary vessels, perhaps to act as a defense against the increased oxidative stress associated with air breathing. However, this possibility remains highly speculative.
In normal lambs, we found no developmental changes in SOD1 protein levels or activity over the first 2 mo of life. Using a rat lung model, Hass and Massaro (11) found that pulmonary SOD1 production was maximal at 1 day after birth and then decreased to adult levels. However, activity was found to increase into adulthood and was attributed to synthesis exceeding degradation (by up to 10%) with no increase in the specific activity of the enzyme. The present study in lambs does not conflict with this rat study since time points studied did not overlap. However, under conditions of increased pulmonary blood flow, SOD1 protein levels were greater in 4- and 8-wk-old shunt lambs than at 2 wk. In vitro studies in endothelial cells and porcine arterioles suggest that shear stress upregulates the expression of SOD1 (7, 8, 28). Since shunt lambs are exposed to increased flow and shear stress, this is likely one mechanism for these changes. However, other potential mechanisms, and the etiology for the lack of a change in associated activity, are unclear and warrant further study.
SOD2 is essential to survival, as deficient mice die within weeks of birth from neurodegenereation, abnormal cardiac development, or mitochondrial damage, while expression is upregulated in models of chronic hypertension (3, 5). In the present study, SOD2 expression increased during postnatal development in both normal and shunt lambs. Initially, at 2 wk of age, protein levels were decreased in shunt lambs, but this reduction was not sustained. In advanced pulmonary vascular disease, a similar decrease in SOD2 protein expression was observed (4). However, it must be noted that SOD2 activity was increased in 2-wk-old shunt lambs, compared with controls, and this activity remained increased over the 2-mo study period. Taken together, our data suggest a developmental upregulation of SOD2 protein and activity in shunts, possibly as an adaptive mechanism to address increases in oxidant species. Due to its subcellular localization in the mitochondrion, SOD2 is thought to be the primary defense to oxidative stress and is very responsive to and upregulated by oxidative stress (5). Shear stress has also been found to induce the expression of SOD2 in vascular endothelial cells (2) and might explain the increase in SOD2 seen in the shunt animals. However, other potential regulatory mechanisms warrant investigation.
Prior studies in the developing human lung (17-wk gestation) indicated that SOD2 expression is limited to the alveolar and bronchial epithelium as well as macrophages (13). However, our immunohistochemical data indicate that in the postnatal lamb lung, there is robust expression of SOD2 in both the endothelium and the smooth muscle layers of pulmonary arteries. Again suggesting that increased postnatal SOD2 expression in the pulmonary vessels acts as a defense against the increased oxidative stress associated with air breathing.
Our data indicate that at 2 wk of age, SOD3 levels are significantly elevated in shunt compared with control lambs but that this increased expression is not maintained over the next 6 wk. Furthermore, no overall changes in SOD3 expression could be detected in the control lambs over the 8-wk period. These modulations in SOD3 expression may be due to alterations in NO signaling as previous studies suggested that NO generation from eNOS can increase SOD3 expression (6) and our previous studies suggest that there is a progressive loss of that NO signaling between 1 and 4 wk of age (19, 21) due to eNOS uncoupling (8, 15). However, further studies will be required to investigate this possibility. In addition, our immunohistochemical analyses indicated that SOD3 is localized only to the smooth muscle layer and is absent from the endothelium. This confirms a previous study that indicated that SOD3 was produced in vascular smooth muscle cells and that it may be involved in endothelium-dependent vasodilation (17). However, other studies indicated that SOD3 is produced by vascular endothelial cells (among others) in the mature lung and is found in extracellular spaces around airway spaces and vessels (18). This suggests that the endothelium of the perinatal lung may be sensitized to increasing superoxide levels. Indeed, depending on the study and anatomical region, the contribution of SOD3 activity varies, and in the aorta SOD3 makes up as much as 70% (18) or as little as 8% (5) of the total SOD activity. Fukai et al. (7) demonstrated an increase in SOD3 protein but not SOD1 in an ANG II-induced hypertension mouse model, while Jung et al. (12) determined that SOD3 was the major antagonist to vascular superoxide. Further research will be required to determine the actual contribution of SOD3 and SOD1 in our lamb model of pulmonary hypertension. Indeed, a caveat for our studies is that we have not directly determined SOD3 activity changes in our model nor determined how the lack of SOD3 expression on the endothelium alters its susceptibility to oxidative stress.
When total SOD activity was quantified, no differences were seen at all time points. As total SOD activity potentially measures the activity of all SOD isoforms present, including extracellular SOD (SOD3), the actual activity of SOD1 may be over- or underestimated. This may serve to explain the developmental increase in SOD1 in 4- and 8-wk shunts without an associated increase in activity and is again suggestive that posttranslational modifications of the SOD system may be occurring in the shunt lambs. Furthermore, our EPR studies comparing shunt and control lambs at 4 wk of age confirmed our previous DHE oxidation data indicating that there are increased superoxide levels in the peripheral lung of the shunt lambs (8). In addition, we found that superoxide levels were also significantly elevated at 2 wk, but not 8 wk, of age in the shunt lambs. Interestingly, both control and shunt lambs exhibited a temporal increase in superoxide levels over the 8 wk studied. This likely represents increased uncoupling of eNOS within the pulmonary vessels as we previously investigated the developmental alteration in NO and superoxide levels from pulmonary artery vessels from fetal and juvenile (4 wk) lambs (16). Our results demonstrated that NOS-derived ROS increase in juvenile pulmonary artery vessels and these are responsible, at least in part, for the vasodilator response at this age. In addition, we found that pulmonary artery endothelial cells (PAEC) isolated from fetal lambs produce significant levels of NO and small amounts of superoxide on shear stimulation, whereas the PAEC isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide (16). These data suggested that eNOS is uncoupled to a certain extent in PAEC during postnatal development and this is supported by the EPR data presented here.
In conclusion, we report that in lambs, developmental changes occur in the enzyme systems responsible for ROS degradation over the first 2 mo of life and that these changes are altered under conditions of abnormally increased pulmonary blood flow. Emerging therapies for pulmonary hypertensive disorders are targeting ROS scavenging systems, as an approach to decrease oxidative stress and improve endothelial function (21). A better understanding of the manner in which the ROS scavengers are altered in disease, and a sensitivity to the duration of disease, will be important to maximize the efficacy of these therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Sharma and A. C. Grobe contributed equally to this work. 
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P < 0.05 vs. shunt at 2 wk.
