Published ahead of print on October 25, 2007, doi:10.1164/rccm.200702-264OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200702-264OC
Gene Transfer of Extracellular Superoxide Dismutase Ameliorates Pulmonary Hypertension in Rats1 Second Department of Internal Medicine, and Departments of 2 Pharmacology and 3 Pathology, School of Medicine, University of Occupational and Environmental Health, Kitakyusyu, Japan; and 4 Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan Correspondence and requests for reprints should be addressed to Hiromi Tasaki, M.D., Ph.D., Second Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku 807-8555, Kitakyushu, Japan. E-mail: h-tasaki{at}med.uoeh-u.ac.jp
Rationale: Pulmonary hypertension (PH) is a life-threatening disease, characterized by vascular remodeling and vasoconstriction. Evidence suggests that oxidative stress may contribute to the pathogenesis and/or development of PH. Objectives: In the present study, we examined whether intratracheal gene transfer of human extracellular superoxide dismutase (EC-SOD) could ameliorate monocrotaline (MCT)–induced PH in rats. Methods: MCT-injected rats were intratracheally administered vehicle (MCT group) or an adenovirus encoding β-galactosidase (Adβgal group) or human EC-SOD (AdEC-SOD group). Measurements and Main Results: After intratracheal gene transfer, EC-SOD was successfully expressed in lung tissue, bronchoalveolar lavage fluid, and plasma. Twenty-eight days after MCT injection, right ventricular systolic pressure and the weight ratio of the right ventricle to the left ventricle plus septum were significantly lower in the AdEC-SOD group (42.50 ± 1.46 mm Hg and 0.453 ± 0.029, respectively) than in the MCT group (59.89 ± 1.61 mm Hg and 0.636 ± 0.022, respectively) or the Adβgal group (61.50 ± 2.61 mm Hg and 0.653 ± 0.038, respectively). Moreover, vascular remodeling and proliferation of vascular smooth muscle cells in pulmonary arteries were markedly suppressed in the AdEC-SOD group. Importantly, 8-isoprostane in lung tissue was also significantly reduced in the AdEC-SOD group. Conclusions: EC-SOD overexpression to the lung ameliorated MCT-induced PH in rats. We suggest that EC-SOD may act as an antioxidant in PH and that increased oxidative stress may be important in the pathogenesis of MCT-induced PH.
Key Words: monocrotaline • oxidative stress • intratracheal gene transfer • epithelial cell
Pulmonary hypertension (PH) is characterized by a progressive elevation of pulmonary arterial pressure, ultimately inducing right ventricular (RV) failure and death with pathological changes in precapillary pulmonary arteries (1, 2). Although bone morphogenetic protein receptor type II was reported as an idiopathic PH–related gene (3), and although clinical use of an endothelin-1 receptor blocker has begun (4), the pathogenesis is not completely understood and available treatments are limited. Studies suggest that increased oxidative stress, such as enhanced production of superoxide anions and other reactive oxygen species (ROS), may contribute to the pathogenesis and/or development of PH (5, 6). Antioxidant enzymes are responsible for regulating oxidative stress in tissues and may play key roles in controlling or preventing these conditions. One family of important antioxidant enzymes in the regulation of oxidative tissue damage is superoxide dismutase (SOD). SOD catalyzes the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Three different isoforms of SOD have been identified in humans: copper–zinc SOD (Cu,Zn-SOD) located in cytosol (7), manganese SOD localized in mitochondria (8), and an extracellular form of Cu,Zn-SOD (EC-SOD) (9). EC-SOD is a secreted protein that exists in the interstitial spaces of tissue and appears to be bound mostly to heparan sulfate proteoglycans via a positively charged COOH-terminal heparin-binding domain. Heparan sulfate proteoglycans exist in the glycocalyx of cell surfaces and in the connective tissue matrix (10, 11). This location allows EC-SOD to efficiently scavenge superoxide anions. Consequently, increased levels of EC-SOD are able to reduce oxidative tissue damage by decreasing superoxide and diminishing the direct and indirect reaction of ROS. In addition, increased EC-SOD can reduce the rapid reaction of superoxide with nitric oxide (NO) and thereby decrease the production of peroxynitrite. It is thus thought that EC-SOD plays a central role in extracellular antioxidant systems (10, 12). It has been shown that in EC-SOD–transgenic mice, lung injury induced by hyperoxia (13), bleomycin (14), or radiation (15) is reduced, and that in EC-SOD–deficient mice, hemorrhage-induced lung injury is increased (16). These findings indicate that EC-SOD may be involved in the pathogenesis of various lung diseases. However, the role of EC-SOD in PH remains unclear. In this study, we examined whether overexpression of human EC-SOD in the lung ameliorates monocrotaline (MCT)-induced PH in rats (an animal model mimicking human PH). We chose to study rats to examine the effects of EC-SOD on MCT-induced PH because the lung tissue and pulmonary arteries of rats have much less endogenous EC-SOD than is found in other species (11, 17).
All animal experimental procedures were reviewed and approved by the Ethics Committee on Animal Experiments and Care of the University of Occupational and Environmental Health (Kitakyushu, Japan).
Recombinant Adenoviral Vectors Replication-deficient adenoviruses encoding human EC-SOD or β-galactosidase driven by a cytomegalovirus promoter were obtained from the Gene Transfer Vector Core (University of Iowa, Iowa City, IA).
Gene Transfer to Sprague-Dawley Rats with MCT Treatment
Expression of β-Galactosidase and EC-SOD Induced by Gene Transfer
Hemodynamic Measurements and Assessment of RV Hypertrophy
Morphometric Analysis of Pulmonary Arteries
Immunohistochemical Analysis On Day 28, proliferation of vascular smooth muscle cell (VSMCs) was analyzed with a monoclonal antibody against proliferating cell nuclear antigen (PCNA; Dako), and in situ apoptosis was assessed by terminal deoxynucleotidyltransferase–mediated dUTP-biotin nick end labeling (TUNEL; Chemicon International/Millipore, Billerica, MA) (22, 23). The numbers of PCNA-positive and TUNEL-positive cells in 10 fields for each section were quantitatively evaluated as a percentage of the total number of cells at a magnification of x400.
Evaluations of Oxidative Product and Endothelial Nitric Oxide Synthase Lung tissue was perfused and rinsed with phosphate-buffered saline (pH 7.4) containing heparin (2 unit/ml) to remove any red blood cells and clots, and then it was homogenized at 4°C. In the lung homogenates, 8-isoprostane and nitrotyrosine were analyzed serially on Days 0, 1, 3, 5, 7, 14, 21, and 28, using a commercial kit for 8-isoprostane (Cayman Chemical Co., Ann Arbor, MI) and nitrotyrosine (Oxis International, Foster City, CA). In addition, Western blot analysis for endothelial NO synthase (eNOS) in the lung was also performed on Day 28 (24).
Statistical Analysis All data are expressed as means ± SEM. Differences between groups were evaluated by analysis of variance followed by the Scheffé post hoc test for multiple comparisons. P values less than 0.05 were considered statistically significant.
Gene Expression Introduced by Gene Transfer Five days after gene transfer, X-Gal staining and immunohistochemical staining with an anti–EC-SOD polyclonal antibody were performed. β-Galactosidase protein (blue nuclei) in Adβgal-transfected lung was expressed diffusely in airway and alveolar epithelial cells, but to a lesser extent in the small pulmonary arteries (Figures 1A and 1B). Although human EC-SOD was not observed in lung tissues in the control, MCT, and Adβgal groups (Figure 1C), the protein (brown nuclei) in AdEC-SOD–transfected lung was expressed diffusely in alveolar epithelial cells (Figure 1D).
EC-SOD Activities and Concentrations in Lung, BALF, and Plasma EC-SOD activities and concentrations in lung tissue, BALF, and plasma were determined on Days 0, 1, 3, 5, 7, 14, 21, and 28 in the AdEC-SOD group. Both EC-SOD activities and concentrations in lung tissue increased after EC-SOD gene transfer, and reached a peak on Day 5 (n = 4 each, 18.77 ± 3.63 U/mg protein and 84.3 ± 29.8 ng/mg protein, respectively), and decreased gradually over 14–21 days (Figures 2A and 2C). Both the EC-SOD activities and concentrations in lung tissue on Day 28 were not significantly different compared with the baseline levels (Figures 2A and 2C). On Day 5, the EC-SOD activities in lung tissue, the EC-SOD concentrations in lung tissue, and that in BALF in the AdEC-SOD group were significantly higher than those in the other groups (n = 4 each, P < 0.001, P < 0.001, and P < 0.001, respectively) (Figures 2B, 2D, and 2E), whereas there was an approximately sixfold increase in the EC-SOD concentrations in plasma in the AdEC-SOD group (n = 4 each, P < 0.001 compared with the other groups) (Figure 2F).
Effects of AdEC-SOD on MCT-induced Pulmonary Hypertension Body weight, systolic blood pressure, and heart rate on Day 28 were measured in conscious rats (Table 1). Body weight was significantly lower in the MCT group than in the control group, but there were no significant differences between the MCT, Adβgal, and AdEC-SOD groups. However, body weight posttreatment and the change in body weight, defined as the difference between pre- and post-treatment body weights, showed a tendency to recover in the AdEC-SOD group compared with the Adβgal group. Systolic blood pressure and heart rate were statistically similar in the four groups (Table 1).
The MCT group (n = 8) had developed severe PH by Day 28, with elevated RVSP (59.89 ± 1.61 mm Hg) (a marker of systolic pulmonary arterial pressure) and an increased RV/(LV + S) weight ratio (0.636 ± 0.022) (an index of RV hypertrophy) compared with the control group (n = 6, 29.73 ± 0.79 mm Hg and 0.254 ± 0.007, respectively). The Adβgal group (n = 6, 61.50 ± 2.61 mm Hg and 0.653 ± 0.038) had increased RVSP and the RV hypertrophy was similar to that of the MCT group. In the AdEC-SOD group (n = 8, 42.50 ± 1.46 mm Hg and 0.453 ± 0.029) development of MCT-induced PH by Day 28 was significantly suppressed (Figures 3A and 3B).
In this study, we examined the effects of AdEC-SOD on the survival of MCT-treated rats. However, the survival rate did not differ between the AdEC-SOD and Adβgal groups during 8 months of follow-up (n = 12, 83% in the AdEC-SOD group; n = 12, 83% in the Adβgal group).
Inhibitory Effects of AdEC-SOD on MCT-induced Medial Wall Thickening and Oxidative Product
We evaluated the effects of AdEC-SOD on VSMCs in the pulmonary arteries. Proliferating cells were detected immunohistochemically with an anti-PCNA antibody, and apoptotic cells were assessed by the TUNEL method. The percentage of PCNA-positive cells in VSMCs was increased on Day 28 in the MCT (26.4 ± 2.0%) and Adβgal (25.0 ± 1.4%) groups, but was significantly decreased in the AdEC-SOD group (7.9 ± 1.3%) (Figure 4E). The percentages of TUNEL-positive cells in VSMCs were similar in the four groups (data not shown). We measured levels of 8-isoprostane and nitrotyrosine in lung tissue from Day 0 to Day 28 to determine the oxidative stress in MCT-induced PH and the role of EC-SOD in modulating oxidative lung damage. In the MCT group 8-isoprostane was significantly increased after Day 5 compared with the control group, and the increased levels in the MCT group were similar to those in the Adβgal group (data not shown). However, 8-isoprostane was markedly reduced on Days 5 and 7, but not on Days 14, 21, and 28, in the AdEC-SOD group (Table 2). Nitrotyrosine on any day was comparable among the four groups. We also analyzed eNOS protein levels. MCT caused a decrease in eNOS protein level in lung tissue, which was significantly reserved by EC-SOD gene transfer (Figure 5).
In the present study, we demonstrated for the first time that intratracheal gene transfer with adenovirus-mediated EC-SOD suppressed MCT-induced PH in rats. This result provides direct support for the notion that increased oxidative stress in the lung plays a crucial role in the pathogenesis of MCT-induced PH. It also shows that diffuse expression of EC-SOD in epithelial cells can be achieved by intratracheal administration of EC-SOD gene–containing fluid, and that this improves RVSP and RV hypertrophy through reduced obstruction in small arteries, which may be connected with inhibition of VSMC proliferation. The lungs are directly exposed to higher oxygen concentrations than most other tissues. Oxidant–antioxidant imbalance is important for the pathogenesis of obstructive lung diseases such as asthma, chronic obstructive pulmonary disease, parenchymal lung disease, and lung malignancies. As for PH, oxidative stress has been shown to be associated with alterations in both endothelin-1 and NO signaling pathways and to be capable of stimulating the proliferation of vascular smooth muscle cells (25). Moreover, Cracowski and coworkers suggested that isoprostanes, which are lipid peroxidation products of arachidonic acid, are increased in patients with PH (26). Thus, increased oxidative stress is thought to contribute to the pathogenesis of PH. It is well accepted that EC-SOD, which is a major extracellular antioxidant enzyme, protects lung tissues against increased oxidative stress. By analysis of mRNA (27) or protein (12, 28), EC-SOD in lung has been found in the extracellular matrix of vessel walls, the bronchial epithelium, the alveolar epithelium, alveolar macrophages, and endothelial cells lining both arteries and veins, and can efficiently eliminate or reduce the amounts of superoxide anions, induced by phagocytic cells or other cell types. Experiments with EC-SOD–transgenic and –deficient mice have shown that EC-SOD is correlated with many pathological conditions in the lung (13–16). Moreover, in newborns with respiratory distress syndrome, EC-SOD is expressed in the arterial intima and endothelium, the metaplastic alveolar epithelium, chondrocytes, and the hyaline membrane (29). These results indicate that EC-SOD may be of potential importance in the regulation of multiple lung diseases; however, no study has addressed the role of EC-SOD in PH. Of the various animal models for PH, we selected the MCT model and rats for this study. The basic mechanism underlying PH induced by MCT, a pyrrolizidine alkaloid, remains to be fully resolved. However, it is reported that MCT induces endothelial injury in pulmonary arteries, followed by VSMC proliferation. It causes pathology resembling human PH and the MCT model is often used as an animal model to mimic human PH (30). Although the EC-SOD protein in most mammals is a tetramer, in the rat it is a dimer and is expressed at much lower concentrations than those in other species including humans (11, 17). Therefore, the present study can confirm direct effects of the human EC-SOD transgene in MCT-induced PH. Gene transfer to blood vessels is usually accomplished by intralumenal delivery, whereas gene transfer to the lung has been performed using intralumenal (31, 32) and intratracheal methods (33, 34). Because the intratracheal method has fewer effects on hemodynamics than the intralumenal method, some studies have concluded that the intratracheal approach is a valuable therapeutic strategy for adenovirus-mediated gene transfer in PH (33, 34). To avoid hypoventilation during and immediately after the gene transfer, we selected the intratracheal method with an artificial ventilation system. In the present study, intratracheal gene administration led to diffuse expression of β-galactosidase or EC-SOD protein in airway and alveolar epithelial cells, but to a lesser extent in the small pulmonary arteries (Figures 1A, 1B, and 1D). Thus, transgenic human EC-SOD expression is achieved in cell-specific locations consistent with physiologic EC-SOD expression (33, 34). Furthermore, significant increases in EC-SOD concentrations in lung tissue, BALF, and plasma demonstrated successful expression of the EC-SOD gene. Expression of EC-SOD in lung tissue peaked on Day 5 and continued at a lower but still significant elevation for 14–21 days thereafter (Figures 2A–2D). On the basis of histologic and biochemical examinations, we speculated that EC-SOD transgene detected in epithelial cells might exert its antioxidative properties against ROS induced by MCT. Moreover, EC-SOD located in endothelial cells might also have some antioxidative function. However, the mechanisms by which genes transferred into the trachea express their proteins in the precapillary arteries remain unclear. Since in situ hybridization could help to resolve this question, we intend to study this in the future. In this study, we measured 8-isoprostane and nitrotyrosine levels in lung tissue, and demonstrated the favorable effect of EC-SOD on increased oxidative stress in MCT-induced PH. The result may support that oxidative stress is involved in MCT-induced PH in rats and that EC-SOD plays an important protective role against increased oxidative stress in PH. We also suggest that eNOS protein was significantly reduced by MCT treatment and recovered by EC-SOD gene transfer (Figure 5). On the basis of these observations, there are several possible mechanisms by which EC-SOD gene transfer might have ameliorated the development of MCT-induced PH. First, it is supposed that MCT may induce inflammation of lung tissue and stimulate both inflammation-related cells, such as neutrophils, and the production of vasoactive substances, such as endothelin-1, angiotensin II, and serotonin. These cells or vasoactive substances are thought to produce superoxide anions that trigger the destruction of lung tissue or vasculature and intravascular platelet aggregation. The diffuse, high expression of EC-SOD on epithelial cells in this study might be the crucial mechanism antagonizing the oxidative process through disproportioning superoxide anions. As supporting evidence, Liu and coworkers reported that serotonin-induced vasoconstriction in pulmonary arteries was induced in EC-SOD–deficient mice and was partially inhibited in EC-SOD–transgenic mice. They concluded that SOD in the extracellular space could be a new pharmacologic strategy for PH therapy (35). The second possibility is that low, but consistent EC-SOD expression in pulmonary arteries might exert antioxidative properties. ROS such as superoxide anions stimulate vasoconstriction or VSMC proliferation through NO reduction, which leads to the pathophysiology of PH. Vascular EC-SOD expression might halt the ROS cascade by disproportioning superoxide anion and maintain NO bioavailability in pulmonary arteries. Vascular EC-SOD could thus reduce the direct or indirect tissue damage induced by ROS. As a third mechanism, our results also suggest that EC-SOD may have reduced the obstructive lesion in precapillary pulmonary arteries by inhibition of VSMC proliferation (Figure 4E). This mechanism differs from that of improved functional vasoconstriction, as described above, and the two might affect each other synergistically. Although it is unclear whether EC-SOD directly targets VSMCs and arrests their overgrowth, it was suggested in an in vitro study that EC-SOD inhibits platelet-derived growth factor–dependent VSMC proliferation (36). Unlike other reports, apoptosis was not induced in our experiments. The reason for this discrepancy is unclear, but it might be derived from differences in the concentration of MCT or EC-SOD. The last mechanism involves transforming growth factor-β, which was reported to be upregulated in MCT-induced PH (37). Marklund reported that transforming growth factor-β reduced EC-SOD, and that gene therapy might restore EC-SOD in MCT-induced PH (38). Further studies are needed to elucidate the roles of cytokines and chemokines in PH and their changes with EC-SOD. There are some limitations to this study. First, we used a relatively low dose of MCT, in accordance with previous reports (39, 40). The MCT dose selected might have resulted in the beneficial effects of EC-SOD transfer on PH. Second, the MCT-induced PH model might not be consistent with PH in humans. The effects of the EC-SOD gene should be confirmed in another PH model, such as clinically relevant hypoxia-induced PH. This study suggests several clinical implications for PH. Because patients with PH may have reduced NOS activity and low NO availability, we could propose intratracheal EC-SOD administration as a feasible therapy. Another possibility is that EC-SOD could be effective in combination therapy with NO inhalation therapy (41). Rebound PH after cessation of NO inhalation has been observed, because of the decreased NOS activity caused by inhaled NO itself. Oishi and coworkers reported that polyethylene glycol–conjugated SOD prevented changes in NOS activity, superoxide, and peroxynitrite, resulting in no increase in pulmonary arterial resistance in rebound PH (42). Thus, ROS scavenging by EC-SOD gene therapy might be helpful in preventing rebound PH with NO inhalation therapy. In the near future, we hope to confirm whether gene transfer with EC-SOD can be an effective therapeutic strategy for human PH in clinical studies. In conclusion, human EC-SOD overexpression in the lung ameliorated the development of MCT-induced PH in rats. We suggest that EC-SOD may act as an antioxidant in PH and that increased oxidative stress may be important in the pathogenesis of MCT-induced PH.
The authors acknowledge the University of Iowa Gene Transfer Vector Core, supported in part by the National Institutes of Health, and the Roy J. Carver Foundation, for viral vector preparation. The authors thank Dr. Yasuhide Nakashima and Dr. Kiyoshi Ozumi for valuable advice on the whole study.
Supported, in part, by a Grant-in-Aid for Scientific Research (no. 18590827) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Tokyo, Japan) and by funding from the Chiyoda Mutual Life Foundation. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org. Originally Published in Press as DOI: 10.1164/rccm.200702-264OC October 25, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form February 16, 2007; accepted in final form October 25, 2007
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AT A GLANCE COMMENTARY
P < 0.01 versus MCT group (analysis of variance followed by Scheffé test).
P < 0.01 versus MCT group (analysis of variance followed by Scheffé test).
