(Chest. 2005;128:594S-596S.)
© 2005
American College of Chest Physicians
Chronic Hypoxia-Enhanced Murine Pulmonary Vasoconstriction*
Role of Superoxide and gp91phox
John Q. Liu, MD;
Efua M. Erbynn, BS and
Rodney J. Folz, PhD
* From the Departments of Medicine and Cell Biology, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham, NC.
Correspondence to: John Q. Liu, MD, Room 341 MSRB, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham, NC 27710; e-mail: john.liu{at}duke.edu
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Abstract
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Chronic hypoxia (CH) is a common cause of pulmonary hypertension (PH). Accumulating evidence suggests that changes in the activity of endothelin (ET)-1 receptors may play an important role in CH-induced PH. After 3 weeks of CH (10% O2) exposure, we found that the isolated intra-pulmonary artery (PA) constrictor response to ET-1 was significantly increased in wild-type (wt) mice. The administration of Cu/Zn superoxide dismutase (SOD) markedly reduced the CH-enhanced maximal PA constrictor response to ET-1, demonstrating the contribution of superoxide to CH-enhanced PA constrictor responses. Using mice that are completely deficient in gp91phox (a subunit protein of the superoxide producing nicotinamide adenine dinucleotide phosphate [NADPH] oxidase), we found that CH-enhanced PA constriction to ET-1 was completely blocked (decreases in mean [± SE] maximal isometric tension from 5.43 ± 0.35 to 3.33 ± 0.19 mN; n = 7; p < 0.01). Using a lucigenin-enhanced chemiluminescence technique to measure superoxide, we found that the 3 weeks of CH significantly increased superoxide levels in PA isolated from wt mice. The addition of ET-1 further increased superoxide production. To demonstrate that the increased chemiluminescence is due to superoxide generation, we added Cu/Zn SOD, which markedly decreased chemiluminescence, demonstrating the specificity of this assay. When gp91phox knockout mice were exposed to CH, they had significantly reduced levels of superoxide compared to CH-treated wt mice. Our results demonstrate that the CH-enhanced PA constrictor response to ET-1 is mediated by NADPH oxidase (gp91phox)-derived superoxide overproduction that may contribute to the pathogenesis of CH-induced PH.
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Introduction
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Chronic hypoxia (CH) is a major cause of pulmonary hypertension (PH) and right ventricular hypertrophy in patients with COPD.6 Some studies234 have reported that CH-induced PH is due to increased vasoconstrictor activities and/or depressed endothelium-dependent vasodilatation along with pulmonary vascular remodeling. Other studies1451011 have suggested that changes in the production of reactive oxygen species, such as superoxide, may mediate these changes. Nonphagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, such as vascular NADPH oxidases, are the major source of reactive oxygen species in the cardiovascular system.7 Recent studies3 have suggested that gp91phox is an important subunit protein that is required for NADPH oxidase activity. However, no studies have examined the role of NADPH oxidase (gp91phox) on CH-increased pulmonary vasoconstrictor activities.
We hypothesized that exposure to CH leads to enhanced gp91phox-dependent superoxide production, which in turn enhances PA constrictor responses. In this study, we used a previously described small-vessel bioassay7 to demonstrate the important role that gp91phox plays in CH-enhanced murine pulmonary artery (PA) constrictor responsiveness.
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Experimental Procedures
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Mice and CH Exposures
Wild-type (wt) C57BL/6 mice and hemizygous NADPH gp91phox knockout (KO) mice (–/Y) [C57BL/6] (The Jackson Laboratory; Bar Harbor, ME) were used for this study. All mice were 10 to 20 weeks of age, and weighed between 22 and 30 g. Mice were housed for 3 weeks in a flow chamber gassed with 10% O2.
PA Ring Contractility Studies
PA rings (internal diameter, 100 to 150 µm) were isolated from the mouse intra-PA and were placed in a small-vessel wire myograph chamber. Changes in PA isometric tension were recorded.7
Measurement of Superoxide
The measurement of superoxide levels in isolated murine PAs were performed using a lucigenin-enhanced chemiluminescence technique.278
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Results
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CH exposure enhanced PA constriction to endothelin (ET)-1 in wt mice (increase in mean [± SE] maximal isometric tension from 3.58 ± 0.23 to 5.43 ± 0.35 mN; n = 6; p < 0.01) [Fig 1
, top, A]. This was reduced by the administration of a superoxide scavenger, Cu/Zn superoxide dismutase (SOD) (150 U/mL) [decrease in maximal isometric tension from 5.43 ± 0.35 to 3.74 ± 0.44 mN; n = 5; p < 0.05]. The CH-enhanced PA constrictor response to ET-1 was completely blocked in PAs isolated from gp91phox KO mice (wt + CH mice, 5.43 ± 0.35 mN; gp91phox KO + CH mice, 3.33 ± 0.19 mN; n = 7; p < 0.01).
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Figure 1. Top, A: effects of ET-1 in pulmonary arteries isolated from wt and gp91phox KO mice following 3 weeks of CH. Cu/Zn SOD (150 U/mL). Changes of contraction tension are expressed as the mean ± SE. Bottom, B: murine PA superoxide production as measured by lucigenin-enhanced chemiluminescence. PAs were isolated from wt and gp91phox KO mice under the conditions indicated. Changes in chemiluminescence signal are expressed as the mean ± SE.
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CH increased PA superoxide production (increase in mean chemiluminescence from 12.0 ± 0.40 to 24.8 ±1.74 relative light units [RLU]/s; n = 6; p < 0.05) [Fig 1, bottom, B] in wt mice that was further increased in the presence of 10 nmol/L ET-1 (37.5 ± 1.8 RLU/s; n = 6; p < 0.01). However, this CH-increased PA chemiluminescence was significantly reduced either by treatment with Cu/Zn SOD (150 U/mL; 18.7 ± 1.17 RLU/s; n = 6; p < 0.01) or in PA isolated from gp91phox KO mice (14.4 ± 0.66 RLU/s; n = 6; p < 0.01).
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Discussion
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This study supports a model of CH-enhanced superoxide formation via a gp91phox-dependent NADPH oxidase pathway. Activation of this pathway leads to enhanced PA vasoconstrictor responses. Enhanced vasoconstrictor response to ET-1, via ETA or ETB receptors, has been documented in various models of CH-associated PH.9 Prior studies have established that CH-induced PH is associated with increased PA vasoconstrictor activity, but the mechanisms by which these PA constrictor responses are modulated remain unclear. In CH-induced PH in rats, ETA or ETB receptor-mediated PA vasoconstriction was increased.9 We show that the exogenous addition of SOD can significantly reduce CH-enhanced vasoconstrictor responses to ET-1 (Fig 1, top, A). This strongly suggests that CH-enhanced vasoconstrictor responses are mediated, at least in part, by an overproduction of superoxide radicals. In PA isolated from gp91phox KO mice, the CH-enhanced vasoconstrictor responses to ET-1 were completely blocked, and the CH-induced superoxide overproduction was markedly reduced (Fig 1, bottom, B), demonstrating that NADPH oxidase (gp91phox) is the major superoxide generator in this model. In conclusion, our studies demonstrate that superoxide overproduction, via NADPH oxidase (gp91phox) following CH, plays a central role in enhancing ET-1-mediated PA vasoconstriction.
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Footnotes
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Abbreviations: CH = chronic hypoxia; ET = endothelin; KO = knockout; NADPH =nicotinamide adenine dinucleotide phosphate; PA = pulmonary artery; PH = pulmonary hypertension; RLU = relative light units; SOD = superoxide dismitase; wt = wild-type
This study was supported, in part, by American Heart Association (Mid-Atlantic Affiliates) Beginning Grant-in-Aid to J.Q. Liu and by NIH grant R01 (HL-64894) to R.J. Folz.
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References
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Abstract
Introduction
