In this study, we tested the hypothesis that extracellular superoxide plays an important role in the development of pulmonary vascular remodeling and pulmonary hypertension secondary to interstitial lung disease, using an established model of bleomycin-induced lung fibrosis. We report that the extensive remodeling in the medial, adventitial, and intimal layers of the pulmonary artery wall after bleomycin treatment was markedly attenuated in mice overexpressing lung EC-SOD. We determined that the bleomycin-induced up-regulation of two genes, Egr-1 and TGF-β (well-established as critical molecules involved in cell proliferation, vascular remodeling, and lung fibrosis), was blocked in the lungs of mice overexpressing EC-SOD. The protection against bleomycin-induced pulmonary vascular remodeling in mice overexpressing EC-SOD was accompanied by protection against late pulmonary hypertension and mortality. These data provide new, convincing evidence for the central role of extracellular superoxide and EC-SOD in the pathogenesis of pulmonary vascular remodeling secondary to lung fibrosis, and moreover provide the rationale to develop new strategies for addressing the extracellular oxidant/antioxidant imbalance in this life-threatening disease process.
In addition to producing lung fibrosis, intratracheal treatment with bleomycin caused severe pulmonary artery remodeling in all three layers of the vessel wall. Although medial wall and adventitial thickening in the pulmonary arteries of mice exposed to bleomycin was previously reported, this evidence is the first, to the best of our knowledge, that bleomycin also results in the formation of occlusive intimal lesions (6
). These neointimal lesions were not apparent until 35 days, and thus the other experiments that concluded by 21 days would not have shown these structural changes. We did not detect medial wall thickening until the 35-day time point, in contrast to reports of medial and adventitial wall thickening at the 14-day time point (6
). Regarding the main methodological difference between our study and previous studies, we both inflation-fixed and perfusion-fixed the pulmonary vasculature in lungs used for morphometric analysis to preserve vessel diameter, which may have accounted for the discrepancy in comparison to lungs processed only with inflation-fixation.
Bleomycin-induced vascular remodeling is a significant finding, because human pulmonary artery hypertension is characterized by severe arteriopathy, including neointimal lesions, whereas classic rodent models of chronic pulmonary hypertension, chronic hypoxia, and monocrotaline only produce changes in the adventitial and medial layers of the vessel, without intimal changes (14
). The predominance of vasoconstriction with limited pulmonary vascular remodeling or inflammation in chronic hypoxia or monocrotaline-induced pulmonary hypertension in mice has raised concerns about the relevance of murine models in understanding human disease (15
). The bleomycin model, which is relatively simple to implement and is well-characterized by extensive inflammation and fibrosis (see Figure E2), therefore provides new opportunities to use genetically engineered mice to test the role of specific molecules in the pathogenesis of severe arteriopathy, with structural changes more similar to those in the clinical setting, particularly for patients with underlying interstitial lung disease.
The key finding in this study is that the overexpression of lung EC-SOD strongly protected against bleomycin-induced pulmonary vascular remodeling. This observation implicates a role for extracellular superoxide in the pathogenesis of vascular remodeling. We observed remodeling predominantly within small pulmonary arteries, which correlates with an important site of increased pulmonary vascular resistance in pulmonary hypertension. Our findings expand on previous work that described the role of EC-SOD in bleomycin-induced fibrosis and in chronic hypoxic pulmonary hypertension. For example, several laboratories showed that a loss of EC-SOD activity exacerbates lung fibrosis, whereas the overexpression of EC-SOD protects against fibrosis attributable to bleomycin, but the effects of EC-SOD activity on bleomycin-induced pulmonary hypertension have not been tested (10
). In addition, we previously reported that the overexpression of EC-SOD protected against chronic hypoxic pulmonary hypertension (5
). We have not tested the impact of EC-SOD on vascular reactivity or inflammation, or used other animal models of pulmonary hypertension. Future investigations in these arenas would likely provide further insights into the role of extracellular superoxide and EC-SOD in the development of pulmonary hypertension (14
). Treatment with bleomycin is associated with more chronic inflammation and oxidative stress than exposure to chronic hypoxia, which enabled us to identify both common and potentially distinct mechanisms contributing to the development of pulmonary vascular remodeling, and in particular, to focus on secondary pulmonary hypertension attributable to lung fibrosis.
Although most studies of bleomycin-induced interstitial lung disease used endpoints related to inflammation and fibrosis, studies of pulmonary hypertension also included measures of cell proliferation, as observed in relevant cell culture models and in vivo
in the early development of pulmonary hypertension. We report a decrease in proliferation in the pulmonary artery wall of EC-SOD TG mice compared with WT mice after treatment with bleomycin. This finding parallels our previous observation that the overexpression of EC-SOD inhibits early (3-day) hypoxia-induced cell proliferation in small pulmonary arteries (5
). In our previous study, we did not observe cell proliferation in the vessels of bleomycin-treated mice at 35 days, and we did not detect proliferation in the pulmonary arteries of chronically hypoxic mice at 35 days. A similar paucity of proliferation was reported in the lesions of humans with pulmonary hypertension, although it is unclear why the proliferation is reduced at the time of extensive vascular remodeling. The observation that lung EC-SOD expression modulates cell proliferation provides potential new avenues of investigation in the study of the pathogenesis of pulmonary hypertension.
We are interested in understanding the mechanisms by which lung overexpression of EC-SOD could protect against bleomycin-induced pulmonary vascular remodeling. We therefore measured the bleomycin-induced up-regulation of two key reactive oxygen species (ROS)–regulated genes that are known to mediate lung fibrosis and pulmonary hypertension, Egr-1 and TGF-β, and report that their up-regulation is blocked in EC-SOD TG mice. Egr-1 is a redox-sensitive transcription factor that is increasingly recognized as a key mediator of cell proliferation, fibrosis, and vascular remodeling. Egr-1 was implicated in the pathogenesis of both chronic hypoxic pulmonary hypertension and lung fibrosis (16
). We previously reported that the overexpression of lung EC-SOD attenuates the early hypoxia-induced up-regulation of Egr-1 (5
). TGF-β has long been recognized as a major molecule responsible for tissue fibrosis and collagen production, including human conditions associated with interstitial lung fibrosis and animal models of bleomycin-induced lung fibrosis. TGF-β induces Egr-1 in both fibroblasts and vascular smooth muscle via a non–SMAD-dependent pathway involving MEK1/2 and ERK1/2, and increases Egr-1 DNA binding activity to the COL1A promoter. Egr-1 was necessary and sufficient to induce COL1A in fibroblasts. In a novel triple-transgenic mouse with inducible overexpression of lung TGF-β concurrent with a knockdown of Egr-1, the absence of Egr-1 protected against the epithelial cell apoptosis and pulmonary fibrosis generated by the induction of TGF-β (17
). Although numerous studies showed that TGF-β induces Egr-1 to mediate fibrosis, reports claim that Egr-1 can also induce TGF-β, leading to a positive feedback loop (18
). Future studies are necessary to determine the mechanism by which extracellular superoxide induces Egr-1 and TGF-β, and how EC-SOD secreted from Type II alveolar epithelial cells protects against vascular remodeling. Secreted EC-SOD may protect the lung and vascular fibroblasts from the increased oxidative stress that leads to the activation of Egr-1. EC-SOD may also modulate the epithelial–mesenchymal transition, which was reported to contribute to bleomycin-induced lung fibrosis (21
). These findings provide strong evidence for extracellular superoxide as a signaling molecule in response to injuries associated with oxidative or nitrosative stress, leading to the modulation of key genes.
Bleomycin is well-known to cause the oxidative stress responsible for lung injury. Bleomycin binds to intracellular iron and reduces molecular oxygen to superoxide and hydroxyl radical, to cause oxidative damage in cellular structures. In addition, bleomycin induces a marked inflammatory response, which further potentiates oxidative damage through leukocyte-derived ROS (22
). Bleomycin-induced lung injury was attenuated by different antioxidant strategies, including N-acetylcysteine + desferoxamine, hydrogen sulfide, edaravone, carnosine, and epigallocatechin-3–gallate, and was also diminished in mice lacking the p47phox subunit (phagocyte oxidase) of nicotinamide adenine dinucleotide phosphate–reduced oxidase, and in mice overexpressing EC-SOD (23
). The phosphodiesterase-5 inhibitor, sildenafil, attenuated injury, along with an inhibition of ROS production (6
). EC-SOD may protect against bleomycin-induced injury by limiting extracellular superoxide–mediated toxicity or by preventing the inactivation of NO, thus preserving NO bioactivity (24
). We found evidence of increased nitrosative stress in the lung in response to bleomycin, and that stress was attenuated in EC-SOD TG mice. We also found that bleomycin-induced increases in eNOS expression at 21 days were lost by 35 days in WT mice, but not EC-SOD TG mice. Thus, in the face of decreasing eNOS expression, the nitrotyrosine formation seen in the WT mice was not likely attributable to increased NO or superoxide derived from uncoupled NOS, and was more likely attributable to increased superoxide from a different source. Inghilleri and colleagues evaluated the in situ
production of oxidative and nitrosative stress, and identified inflammatory cells as well as airway epithelium as the major sites of production of ROS and NO, although Inghilleri and colleagues did not elucidate the source of ROS production (22
). Bleomycin-induced pulmonary hypertension is also attenuated by sildenifil, which may also be mediated by its effect on NO bioactivity through its preservation of the half-life of cyclic guanosine monophosphate (cGMP) (25
). In a neonatal model of bleomycin-induced pulmonary hypertension and bronchopulmonary dysplasia, chronic treatment with inhaled NO attenuated right ventricular hypertrophy and medial wall thickness (13
). Ultimately, the specific contributions of reactive oxygen and nitrogen species, particularly in the extracellular compartment, to bleomycin-induced pulmonary vascular remodeling, and these species' potential role in the up-regulation of redox-sensitive genes, remain to be determined.
Along with pulmonary vascular remodeling, we found that WT mice developed pulmonary hypertension by 7 days, which persisted for the 35-day study period. In mice overexpressing EC-SOD, the most striking finding was a significant attenuation in pulmonary hypertension at the 35-day time point, which coincided with the protection against vascular remodeling. The progressive increase in pulmonary hypertension in WT mice was also blunted in EC-SOD TG mice. Both vasoconstriction and vascular remodeling can contribute to pulmonary hypertension, and pulmonary hypertension in response to bleomycyin was found to be mediated, at least in part, by vasoconstriction. For example, McNamara and colleagues reported that treatment with a Rho kinase inhibitor acutely reversed the elevated pulmonary vascular resistance in bleomycin-treated neonatal rats (13
), and Hemnes and colleagues showed how a phosphodiesterase inhibitor that lowered pulmonary vascular resistance improved bleomycin-induced pulmonary hypertension at 14 days (6
). Thus EC-SOD does not appear to protect against the early component of bleomycin-induced pulmonary hypertension attributable to vasoconstriction before the onset of vascular remodeling. An improvement in remodeling without an effect on pulmonary hemodynamics was observed in other settings. For example, treatment with rosiglitazone prevented chronic hypoxia-induced pulmonary vascular remodeling, but failed to lower mean pulmonary artery pressures (26
). In the clinical arena, patients with pulmonary hypertension who do not respond to vasodilator therapy have worse outcomes, because current clinical therapies do not target arteriopathy, providing strong evidence for the critical role of vascular remodeling in disease outcomes. Nonetheless, EC-SOD would not be expected to exert effects on vascular tone in our study, because other models of vascular dysfunction indicated that EC-SOD can modulate vascular tone through the regulation of nitric oxide bioactivity (24
). EC-SOD may also modulate pulmonary vascular tone in part by preserving the up-regulation of eNOS and thus NO production in response to bleomycin. Moreover, the site of EC-SOD overexpression by Type II cells in this particular strain of mouse may provide protection at the site of remodeling, but may not be situated to protect completely against vasoconstriction. WT mice may also exhibit more cardiac dysfunction compared with EC-SOD TG mice, so that similar right ventricular systolic pressure in EC-SOD TG mice might reflect improved pulmonary vascular resistance. Several studies demonstrated that intratracheal bleomycin in WT mice results in a decrease in cardiac output (9
). We did not measure cardiac function in this study. However, if bleomycin produces direct cardiotoxicity, we would not expect lung-directed EC-SOD overexpression to protect against cardiac damage. The WT mice dying at 21 and 35 days may have manifested worse pulmonary hypertension, leading us to underestimate the protection by EC-SOD overexpression against pulmonary hypertension at these later time points. Whereas this study focused on the marked protection against remodeling, future studies are needed to address directly the effects of EC-SOD on vascular reactivity in models of pulmonary hypertension, and to determine the contributions of the site of EC-SOD expression on vascular responses.
Along with marked protection against vascular remodeling and late pulmonary hypertension, we report that mice overexpressing lung EC-SOD also had a significant decrease in mortality compared with WT mice. The reported mortality after bleomycin treatment in WT mice has varied in the literature, and is most likely to be determined by dose, length of observation, and method of lung delivery, with administration via tracheotomy associated with early death in contrast to intubation and intratracheal instillation, as performed in this study (6
). Our observed mortality in both mouse strains was much higher than reported by Bowler and colleagues, who cited a 3% overall mortality at 14 days in both WT and EC-SOD TG mice (11
). The analysis by Bowler and colleagues (11
) included mice exposed for only 3, 7, 10, and 14 days, and we similarly found low mortality in both strains when we only examined mice exposed to early time points up to 14 days after exposure, with mortality almost exclusively occurring later. The cause of mortality is likely multifactorial, because previous studies documented severe lung fibrosis with impaired gas exchange, impaired cardiac output, and pulmonary hypertension, each of which can affect outcomes (6
). The observation that EC-SOD overexpression protected against pulmonary vascular remodeling and mortality, with little to no impact on early pulmonary hypertension before the development of significant pulmonary vascular remodeling, suggests that pulmonary vascular remodeling is a major factor in the morbidity and mortality of this disease process, and is mediated by an imbalance in the production of extracellular superoxide and scavenging by EC-SOD.
In conclusion, we tested the hypothesis that extracellular superoxide generated in the lung after the administration of bleomycin contributes to pulmonary hypertension and pulmonary vascular remodeling in this model of lung fibrosis. Wild-type mice treated with intratracheal bleomycin developed significant pulmonary vascular remodeling with medial wall thickening, increased COL1A1 mRNA expression, adventitial collagen deposition, proliferation in the vessel wall, and ultimately, occlusive intimal lesions. Two key genes known to contribute to vascular remodeling and lung fibrosis, Egr-1 and TGF-β mRNA, increased in lung homogenates 7 days after the administration of bleomycin. WT mice also developed elevated right ventricular systolic pressures by 7 days that persisted for 35 days, with a mortality rate approaching 50% by 35 days. In contrast, the overexpression of lung EC-SOD significantly protected against bleomycin-induced remodeling in the adventitial, medial, and intimal layers of the pulmonary artery wall, and prevented the up-regulation of Egr-1 and TGF-β. EC-SOD TG mice were protected against late pulmonary hypertension, and achieved a dramatic improvement in mortality. These data provide new insights into the role of EC-SOD and extracellular superoxide in the pathogenesis of pulmonary hypertension, and identify new potential targets of superoxide that can mediate pulmonary vascular remodeling. This study provides a strong foundation for developing novel therapeutic approaches to restore extracellular oxidant/antioxidant balance in the lung and pulmonary circulation, to protect against the pulmonary hypertension complicating interstitial lung diseases.