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Am J Physiol Lung Cell Mol Physiol 290: L2-L10, 2006. First published August 5, 2005; doi:10.1152/ajplung.00135.2005
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Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox)

John Q. Liu,1 Igor N. Zelko,1 Efua M. Erbynn,1 James S. K. Sham,2 and Rodney J. Folz1

1Division of Pulmonary, Allergy, and Critical Care Medicine, Departments of Medicine and Cell Biology, Duke University Medical Center, Durham, North Carolina; and 2Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 24 March 2005 ; accepted in final form 29 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.

chronic hypoxia; normoxia

SUPEROXIDE HAS BEEN RECOGNIZED as an important factor in the regulation of vascular tone and function (12, 49). Whereas several molecular complexes, including the mitochondrial electron transport chain (METC), cytochrome P-450, and xanthine oxidase can generate superoxide, both METC and NADPH oxidases have been documented to be important sources of superoxide production in the vasculature (24, 40, 53, 59, 61). NADPH oxidases are multisubunit proteins containing membrane and cytoplasmic components. Membrane-bound cytochrome b558, for example, consists of gp91phox and p22phox subunits. These subunits associate with the additional cytoplasmic subunits p47phox and p40phox and the small monomeric G protein Rac. Gp91phox and Rac have both been found in endothelium and vascular smooth muscle cells (30, 47). In mice with a targeted deletion of the gp91phox subunit, superoxide overproduction is markedly reduced in isolated murine aortas after angiotensin II infusion (48, 56) as well as in coronary arteries following hypoxia-reoxygenation (37), indicating an essential role gp91phox plays in the production of superoxide during these processes. Interestingly, despite superoxide production being severely inhibited, Archer et al. (2) have reported that hypoxic pulmonary vasoconstriction, and other elements of oxygen sensing, are preserved in gp91phox knockout (KO) mice. Under basal conditions, cellular concentrations of superoxide are tightly regulated and appear to be maintained at low levels by the enzymatic dismutation of superoxide dismutases (SODs). However, its production can be greatly augmented under pathophysiological conditions to elicit a wide variety of biological responses, many of which are currently being better defined and characterized (12, 26, 35).

Exposure to chronic hypoxia (CH) is known to induce pulmonary arterial hypertension (PAH), characterized by profound vascular remodeling and enhanced vasomotor tone and responsiveness (15, 28, 31). Recent studies suggest that superoxide overproduction may be associated with pulmonary vascular remodeling changes seen after CH-induced PAH (25, 41). In rats, CH enhances pulmonary artery (PA) vasoconstrictor responses to 5-hydroxytryptamine (5-HT) (38, 39). Under normoxic conditions, 5-HT-induced pulmonary vasoconstriction is partly mediated by increased superoxide production (35). Administration of SOD or the NADPH oxidase inhibitor diphenyleneiodonium significantly attenuates acute hypoxic pulmonary vasoconstriction (36, 62). Thus CH-associated increases in superoxide formation may interact with and modulate agonist-mediated PA vasoconstrictor responses.

In this study, we test the hypothesis that increased superoxide production, via a vascular gp91phox-dependant NADPH oxidase, leads to enhanced intrapulmonary artery (IPA) vasoconstrictor responsiveness, IPA smooth muscle remodeling, right ventricle (RV) hypertrophy, and PAH. To test this hypothesis, we examined murine IPA superoxide levels, IPA constrictor activity, RV pressure, and histology of murine lungs and hearts obtained from wild-type (WT) and gp91phox KO mice under both normoxic and CH conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CH exposure. Male WT C57BL/6 mice and male NADPH gp91phox KO mice (–/Y, hemizygous, C57BL/6, stock no. 002365) were obtained from Jackson Laboratories (Bar Harbor, ME). Before each experiment, gp91phox KO mice were genotyped by PCR. All mice were 10–20 wk of age and weighed between 20 and 30 g. Mice were housed in an airflow chamber (gassed with 10% O2) for 3 wk. Cage maintenance required no more than 10-min exposure to room air daily. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg body wt). The chest cavity was opened, and the lungs were rapidly removed and placed into Krebs-Ringer bicarbonate solution (KRBS). The KRBS contained (in mM): 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaCO3, and 10 glucose. All protocols and procedures in this study were approved by the Duke University Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines.

Measurement of murine IPA superoxide. The measurement of superoxide anion levels in isolated murine IPA was accomplished using a lucigenin (bis-N-methyacidinium nitrate)-enhanced chemiluminescence technique (5, 21) and detected using a scintillation counter (luminometer) with two photomultiplier tubes (Biolumat LB 9506; Berthold, Wildbad, Germany). Lucigenin (5 µM) was dissolved in KRBS, as this concentration has been shown to accurately reflect superoxide levels (35, 55). The total volume of lucigenin-buffer solution was 1 ml. After the background chemiluminescence signal had stabilized for 5 min, photon emissions from the isolated murine PA were continuously recorded. The specific chemiluminescence signal (emitted light) was recorded as relative light units per second (RLU/s).

Detection of {alpha}-actin. Isolated murine IPAs (~100–150 µm in intraluminal diameter and 2 mm long) were homogenized in 100 µl of RIPA lysis buffer (0.5% deoxycholate, 1.0% Nonidet P-40, and 0.1% SDS in PBS). After incubation on ice for 30 min, the cell lysate was centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was used for Western blot analysis. Proteins from PA lysate (5 µl) were resolved on SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, and probed with a mouse monoclonal antibody against {alpha}-actin (Sigma, St. Louis, MO). The immunoblot was visualized using ECL Plus reagent (Amersham, Piscataway, NJ), and intensities were quantified using a Typhoon 9400 imager (Amersham).

Measurement of murine RV pressure. Mice were anesthetized with pentobarbital sodium, the tracheas were cannulated, and the lungs were ventilated with 10% O2 or room air at a tidal volume of 0.2 ml and a rate of 75 respirations/min. An incision was made in the abdomen, and the diaphragm was visualized. A 23-gauge needle connected to a pressure transducer was inserted through the diaphragm into the RV. The pressure was measured and recorded by a Horizon 2000 system (Mennen Medical, Clarence, NY). RV puncture was verified by postmortem examination.

Histological studies of murine lungs and heart. Murine lungs and heart were isolated from WT mice and gp91phox KO mice under normoxic and CH conditions. The left lung and heart were fixed (lung was fixed by PA instillation) with 10% formalin. The lungs and heart were then sectioned (3 µm thick) and stained with hematoxylin and eosin. The lung sections were examined and digitally photographed under a light microscope (x600). Pulmonary arterioles (~60 µm in external diameter) were randomly selected from each lung section. The diameter and wall thickness of the pulmonary arterioles and heart were measured using Image-Pro plus software (Silver Spring, MD).

IPA ring contractility studies. IPA rings, 100- to 150-µm internal diameter and 1.9 ± 0.2 mm long, were isolated from the IPA (3rd-4th generation) using a dissection microscope. Each IPA was placed in a small vessel wire myograph chamber (DMT, Aarhus, Denmark) and mounted by threading two steel wires into the lumen and securing the wires to two supports. One support was then attached to a micrometer allowing for the control of ring circumference; the other support was attached to a force transducer for measurement of isometric tension. The whole preparation was kept within a chamber filled with KRBS (pH 7.35–7.45), bubbled with 10% O2 (CH) or 20% O2 (normoxia), 5% CO2, and balance N2, and maintained at 37°C. A Plexiglas cover was placed over the chamber to control oxygen tension over the superfusate. The temperature and PA tension were recorded using a data acquisition and analysis program (DMT).

Initially, isolated murine IPA rings were allowed to equilibrate in the chamber for 10–15 min at an initial tension of 0 mN (1 g = 4.905 mN). Tension was then increased to 5 mN in 2.5-mN steps at 4- to 5-min intervals and held constant thereafter. In IPA isolated from WT mice, the resting tension and length relationship was not significantly different under normoxic and CH conditions. Preliminary experiments were performed to optimize resting tension for maximal constrictor response. To assess vascular viability, the IPA was treated with 60 mM KCl, and tension was recorded. After repeated washing with KRBS, the IPA was exposed to U-46619 (thromboxane A2 agonist, 0.01 µM), followed by acetylcholine (1 µM), and the resulting tension was recorded. After these responses stabilized for 5–10 min, the agonists were washed out of the myograph chamber with KRBS.

RNA isolation and RT-PCR. Total RNA was isolated and purified from PAs using RNAqueous-Micro Kit (Ambion, Austin, TX) and treated with DNase I to remove trace amounts of contaminating genomic DNA. The synthesis of single-stranded DNA from RNA was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer.

The forward (5'-ACT GGG CTG TGA ATG AAG G -3') and reverse (5'-CCT CCG AAT GGT TTT GGT AG-3') primers were designed to amplify mouse gp91phox cDNA. PCR reactions were performed using SureStart Taq Polymerase (Stratagene, La Jolla, CA) at 95°C for 5 min, and 37 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min. The amplified DNA (589 bp) was separated on 1.5% agarose gel and visualized by ethidium bromide staining. Murine GAPDH mRNA was amplified as an internal control.

Reagents. 5-HT and bovine CuZn-SOD were purchased from Sigma. U-46619 was purchased from Cayman Chemical (Ann Arbor, MI). Concentrations expressed are the final molar concentrations in the perfusate.

Data analysis. 5-HT- and U-46619-induced murine PA vasoconstrictions are expressed as increases in tension (mN) from baseline. T-tests and analysis of variance were used for statistical comparisons, taking repeated measures into account when appropriate. When analysis of variance yielded a significant F-ratio, least significant differences were calculated for pairwise comparison between means. P values ≤0.05 indicated statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CH increases IPA superoxide production. Superoxide levels were measured in isolated murine IPA using a lucigenin (bis-N-methyacidinium nitrate)-enhanced chemiluminescence technique (5, 35–37). Three weeks of CH (10% O2) significantly increased superoxide levels in IPA isolated from WT mice [increases in chemiluminescence signal from 12.0 ± 0.4 (normoxia controls) to 24.8 ± 1.7 RLU/s, n = 6, P < 0.001, Fig. 1]. The addition of 5-HT (1 µM) caused further increases in superoxide production (to 41.3 ± 2.9 RLU/s, n = 6, P < 0.001, Fig. 1). The increase in chemiluminescence in CH IPAs was due specifically to superoxide as the addition of CuZn-SOD (150 U/ml) markedly decreased chemiluminescence signal (from 41.3 ± 2.9 to 16.3 ± 1.2 RLU/s, n = 6, P < 0.001, Fig. 1). To determine whether the gp91phox-associated NADPH oxidase is responsible for the CH-enhanced superoxide generation, gp91phox KO mice were exposed to 3 wk of CH. In contrast to WT mice, IPA isolated from the CH-treated gp91phox KO mice showed no significant increase in superoxide production compared with normoxic treated gp91phox KO mice (12.3 ± 0.5 vs. 14.4 ± 0.7 RLU/s, Fig. 1). IPA isolated from gp91phox KO mice exposed to normoxia and CH showed significantly increased chemiluminescence after 5-HT treatment (18.67 ± 0.71 and 17.4 ± 0.6 RLU/s, P < 0.001). However, the magnitude of the increase was substantially lower than that of WT mice only in the CH plus 5-HT group of mice (P < 0.001). As in WT mice, the addition of CuZn-SOD reduced superoxide levels to basal levels.



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  		Fig. 1. Murine intrapulmonary arteries (IPA) superoxide production as measured by lucigenin-enhanced chemiluminescence. IPAs were isolated from wild-type (WT) and gp91phox knockout (KO) mice under normoxic and chronic hypoxia (CH) conditions to which were added 5-hydroxytryptamine (5-HT; 1 µm) or CuZn-superoxide dismutase (SOD) (150 U/ml). Changes in chemiluminescence signal are expressed as means ± SE; n = number of animals. Inset shows the genotyping results (RT-PCR) of gp91phox and GAPDH genes in IPA from WT and gp91phox KO mice. RLU/s, relative light units per second.

 
The increase in NADPH oxidase-dependent ROS production in CH IPA could be due to an increase in oxidase-specific activity, an upregulation of enzyme levels, or vascular smooth muscle remodeling (smooth muscle cellular proliferation and/or hypertrophy). To test for these possibilities, levels of the NADPH oxidase subunit mRNAs, in both normoxic and CH IPAs, were quantified used real-time RT-PCR. Under normoxic conditions, the relative expression of mRNA for gp91phox, p22phox, p40phox, p47phox, p67phox, Rac1, and Rac2 in IPA were 0.78 ± 0.25, 1.07 ± 0.06, 0.93 ± 0.24, 0.88 ± 0.11, 1.12 ± 0.42, 0.95 ± 0.04, and 0.99 ± 0.31, respectively. After CH, the relative expression of mRNA for gp91phox, p22phox, p40phox, p47phox, p67phox, Rac1, and Rac2 in IPA were 0.91 ± 0.17, 1.23 ± 0.18, 1.31 ± 0.05, 1.45 ± 0.48, 1.01 ± 0.29, 1.15 ± 0.43, and 0.93 ± 0.37, respectively (Fig. 2). There were no significant differences between normoxic and CH conditions, suggesting that CH did not increase NADPH oxidase subunit mRNA levels. Second, the increases in chemiluminescence seen after CH treatment were not simply due to an increased amount of smooth muscle due to CH-induced vascular remodeling. Here, we normalized IPA chemiluminescence to {alpha}-actin vascular smooth muscle levels of the IPA by Western blot analysis. We found that CH exposure significantly increases {alpha}-actin vascular smooth muscle content compared with normoxia (1.36 ± 0.09 vs. 1.12 ± 0.12, P < 0.05); however, even after normalizing the chemiluminescence output to {alpha}-actin vascular smooth muscle, CH-exposed IPA still show a significant increase compared with normoxic IPA.



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  		Fig. 2. Relative expression (RT-PCR) of NADPH oxidase subunits in IPA isolated from WT mice under normoxic and CH conditions. Data are expressed as means ± SE. All NADPH oxidase subunits are detected in IPA isolated from 3 WT mice under normoxic and CH conditions, respectively (except Rac2 and p67phox, which are derived from 12 WT mice under normoxic and CH conditions, respectively).

 
gp91phox KO abolishes CH-induced pulmonary hypertension. Under normoxic conditions, mean RV pressure was 10.4 ± 0.6 mmHg (n = 8) in WT mice (Fig. 3). After 3 wk of CH (10% O2) exposure, the mean RV pressure increased to 33.3 ± 2.0 mmHg (n = 6, P < 0.001, Fig. 3). However, gp91phox KO mice were resistant to CH-induced RV hypertension, showing a mean RV pressure of 13.4 ± 0.3 mmHg after 3 wk of CH treatment (n = 4, P < 0.001, Fig. 3). Normoxic gp91phox KO mice had RV pressures (11.3 ± 0.4 mmHg, n = 7) comparable to that of WT mice (Fig. 3).



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  		Fig. 3. Measurements of murine mean right ventricle (RV) pressure in WT and gp91phox KO mice under normoxic and CH conditions. RV pressure is expressed as the mean right ventricular pressure ± SE; n = number of animals.

 
Pulmonary hypertension is characterized by vascular smooth muscle remodeling and is associated with a decrease in the diameter of small resistance arteries and arterioles (15, 19, 28). The effects of CH on smooth muscle growth/remodeling in small resistant PAs in WT and gp91phox KO mice were compared by analyzing the wall thickness of PAs with external diameters of ~60 µm (Fig. 4). PAs from WT mice exposed to 3 wk of CH showed a significant increase in vessel wall thickness (Fig. 4) compared with normoxic WT mice (8.0 ± 0.4 µm vs. 5.0 ± 0.3 µm, P < 0.001). Additionally, after normalizing vessel wall thickness to a percent of external diameter (% of external diameter), we also found the vessel wall thickness to be significantly higher in the WT plus CH treatment vs. WT plus normoxia (27.8 ± 1.0% vs. 17.8 ± 1.0, P < 0.001). Consistent with our previous RV pressure studies, we found that CH-induced increases in vascular wall thickness were completely blocked (15.9 ± 1.4%, Fig. 4) in mice lacking gp91phox.



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  		Fig. 4. Morphometric studies of murine intrapulmonary arterioles. Murine lungs were isolated from WT and gp91phox KO mice after normoxic and CH conditions, and inflation was fixed in 10% formalin. Lungs were sectioned, examined under light microscopy, and digitally analyzed at x600 magnification. The vascular wall thickness as a % of external diameter was measured and expressed as means ± SE; n = number of vessels.

 
RV hypertrophy is often used as a metric for pulmonary hypertension (15, 19, 28). In this study, RV hypertrophy was quantified by measuring the ratio of the RV wall thickness to the combined thickness of the left ventricular (LV) wall plus septum (LV + S). CH caused significant RV hypertrophy in WT mouse heart (increase in wall thickness from 0.40 ± 0.05 to 0.70 ± 0.05 mm, P < 0.001) without affecting LV wall thickness (Fig. 5). After normalizing to LV + S, the RV/(LV + S) ratio also showed RV hypertrophy in the WT CH-treated group (0.14 ± 0.01 vs. 0.25 ± 0.01, P < 0.001). In contrast, CH-induced RV hypertrophy was completely blocked in mice lacking gp91phox [RV wall thickness 0.43 ± 0.01 mm; the RV/(LV + S) 0.15 ± 0.01, P < 0.001], with no differences compared with the normoxic WT group (Fig. 5).



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  		Fig. 5. Morphometric studies of murine RV hypertrophy: murine hearts were isolated from WT and gp91phox KO mice after normoxic and CH conditions and were fixed in 10% formalin. Heart tissues were sectioned, examined under light microscopy, and digitally analyzed at x30 magnification. The RV and left ventricular (LV) wall thickness was measured and is expressed as means ± SE; n = number of animals. S, septum.

 
gp91phox KO abolished CH-enhanced vasoconstrictor responses of isolated PA. Under normoxic conditions, 5-HT (0.1–10 µM) causes a concentration-dependent vasoconstriction (maximal constriction tension, Emax = 2.97 ± 0.20 mN, EC50 = 0.27 ± 0.05 µM, Fig. 6A) in IPA obtained from WT mice. However, after 3 wk of CH conditions, IPA showed a markedly enhanced, concentration-dependent, vasoconstrictor response to 5-HT (Emax increased to 4.86 ± 0.43 mN, P < 0.01, and EC50 = 0.09 ± 0.01 µM, P = 0.005 vs. normoxic group, Fig. 6A). Additionally, IPA from CH-treated mice showed enhanced vasoconstriction to 0.01 µM U-46619 (Emax increased from 2.83 ± 0.19 to 4.74 ± 0.42 mN, P < 0.01, Fig. 6B). Previously, we have demonstrated that extracellular superoxide enhances 5-HT-induced vasoconstriction in IPA under normoxic conditions (35). To determine whether superoxide contributes to CH-enhanced PA constriction, the enzymatic scavenger of superoxide, CuZn-SOD, was used. The addition of CuZn-SOD (150 U/ml) significantly reduced CH-enhanced PA constrictor responses to 5-HT (decrease in Emax from 4.86 ± 0.43 to 3.37 ± 0.35 mN, P < 0.01, EC50 = 0.08 ± 0.01 µM, P = 0.007 vs. CH group, Fig. 6A) and to U-46619 (decrease in Emax from 4.74 ± 0.42 to 3.97 ± 0.33 mN, P < 0.01, Fig. 6B).



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  		Fig. 6. Vasoconstriction dose-response curves of IPAs to 5-HT (A) and U-46619 (B). Pulmonary arteries were isolated from WT mice under normoxic and CH conditions. Where indicated, CuZn-SOD (150 U/ml) was added before agonist. Changes in contraction tension (mN) are expressed as means ± SE; n = number of animals.

 
We also examined vasoconstrictor responses of IPA from gp91phox KO mice. Under normoxic conditions, the vasoconstrictor responses to 5-HT were similar to IPA isolated from WT mice (Emax = 2.99 ± 0.26 mN, and EC50 = 0.18 ± 0.02 µM, P = 0.558 vs. WT normoxic group, Fig. 7A). After 3 wk of CH, the CH-enhanced vasoconstrictor responses to 5-HT were significantly attenuated (Emax of 3.63 ± 0.26 mN, P < 0.01 vs. WT + CH group, EC50 0.20 ± 0.09, P < 0.05 vs. WT + CH group, Fig. 7A) in IPA from CH-treated gp91phox KO mice.



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  		Fig. 7. Vasoconstriction dose-response curves of IPAs to 5-HT (A) and U-46619 (B). Pulmonary arteries were isolated from WT and NADPH oxidase gp91phox KO mice under normoxic and CH conditions. Changes in contraction tension (mN) are expressed as means ± SE; n = number of animals.

 
Furthermore, the CH-enhanced vasoconstriction to U-46619 was also markedly reduced in IPA from CH gp91phox KO mice (Emax of 3.46 ± 0.31 mN, P < 0.05, Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study are 1) chronic hypoxia increases superoxide production from IPAs, and this process was dependent on the presence of a functional gp91phox containing NADPH oxidases; 2) disruption of NADPH oxidases by gp91phox gene KO completely abolished chronic hypoxia-induced pulmonary hypertension and vascular remodeling; and 3) gp91phox gene KO significantly attenuated 5-HT-induced superoxide production and enhancement of 5-HT-induced vasoconstrictor responses in chronic hypoxic IPAs. As far as we are aware, these are the first studies that directly demonstrate a major role for NADPH oxidases in the development of chronic hypoxic PAH.

CH increases IPA superoxide production via NADPH oxidases. Reactive oxygen species (ROS), including superoxide and H2O2, are known to play important roles in signal transduction in pulmonary vasculatures. Recent evidence supports the idea that acute hypoxia causes the overproduction of ROS, which seems to contribute to hypoxic pulmonary arterial vasoconstriction (1, 36, 40, 51, 62). In pulmonary arterial smooth muscle cells and endothelial cells, the source of increased ROS during hypoxia appears mediated primarily through the mitochondrial electron transport chain and xanthine oxidase, respectively (36, 40, 54, 59). Similar to these studies, superoxide specifically has been implicated in both a rat and mouse model of CH-induced PA smooth muscle remodeling and pulmonary hypertension (25, 41). When measured by electron spin resonance, ROS production was significantly higher in lung homogenate of chronic hypoxic rats (41), and inhibition of ROS production has been shown to attenuate hypoxic pulmonary hypertension (16, 25). There are many potential biochemical sources of superoxide, including the mitochondria (14, 40), NADPH oxidase (6, 40, 46), xanthine oxidase (25, 42), cytochrome P-450 (50), cyclooxygenases, and endothelial nitric oxide synthase (27, 63) as well as several different types of vascular and nonvascular cells in the lung, which can contribute to their generation. What is not known is whether the ROS overproduction seen in CH is related specifically to pulmonary vasculature, and, if so, which pathway is the major source of ROS production. In the present study, we demonstrate that exposure to CH leads to superoxide overproduction in IPAs obtained from these mice (Fig. 1). More importantly, this CH-enhanced superoxide overproduction was completely blocked in IPA isolated from gp91phox KO mice (Fig. 1), suggesting that a gp91phox-dependent NADPH oxidase is the major source of superoxide production in the chronic hypoxic pulmonary vasculatures. As we have reported previously (35) and again show in Fig. 1, 5-HT increases superoxide production in IPA via a gp91phox-independent pathway, whereas CH-increased superoxide production occurs via a NADPH oxidase pathway that is dependent on gp91phox, suggesting that 5-HT and CH utilize different mechanisms to activate the NADPH oxidase pathway.

NADPH oxidase activity has been identified in both endothelium and vascular smooth muscle (6, 30, 33, 40). It consists of a membrane-bound cytochrome b558 (itself composed of gp91phox and p22phox subunits) and cytoplasmic components such as p47phox, p40phox, and the small monomeric G protein Rac (30, 43). It can be activated by mechanical forces such as laminar and oscillatory shear stress (6, 40, 46) and by lipids, cytokines, and hormones, including angiotensin II, PDGF, TNF-{alpha}, thrombin, and lactosylceramide (3, 18, 23, 29, 58). Moreover, the NADPH oxidase within pulmonary vasculature has been implicated in pathophysiological processes (6, 35, 40, 46). For example, in pulmonary hypertension of fetal lambs, increased superoxide production in PA is associated with high levels of p67phox, a subunit of the NADPH oxidase complex (6). Hence, the overproduction of ROS from NADPH oxidases in IPAs of CH mice could be related to an increase in NADPH oxidase activity induced by hypoxia, mechanical and circulating factors, and/or by an upregulation of NADPH oxidase subunit expression. Although CH exposure increased IPA smooth muscle {alpha}-actin expression (consistency with CH-increased IPAs wall thickness), we did not find any effects on mRNA levels of the major NADPH oxidase subunits in IPA, such as gp91phox, p22phox, p40phox, p67phox, Rac1, and Rac2. These findings along with our data showing that superoxide overproduction in CH WT IPAs was significantly limited in IPAs isolated from gp91phox KO mice suggest that the overproduction of ROS after CH likely comes from enhanced NADPH oxidase activity in IPAs of CH mice.

NADPH oxidase and pulmonary vascular remodeling. The pathophysiology of CH-induced PAH is very complex and likely involves multiple factors. Rho-kinase (17), hypoxia-inducible factor-1 or -2{alpha} (8, 64), vascular endothelial growth factor-b (57), 5-HT1B or 2B receptors and 5-HT transporter (28, 31, 39), ET-1 and its receptor(s) (11, 13), prostacyclin (19), heparin (22), calcitonin gene-related peptide (4), serine elastase (65), nitric oxide (52), and store- and receptor-operated Ca2+ channels (34) have all been implicated to play an important role in CH-induced PAH. However, the relative importance and integration of each these factors in the pathogenesis of CH-induced PAH remain less well understood. We found that CH-induced increases in pulmonary arterial pressure (as indexed by mean RV pressure), vascular smooth muscle thickening, and right heart hypertrophy were completely blocked in gp91phox KO mice. These results suggest unequivocally that superoxide produced from gp91phox-dependent NADPH oxidase plays a key role in this physiological adaptation process. A hallmark feature of chronic hypoxic pulmonary hypertension is the profound vascular remodeling with medial and adventitial thickening in small PAs due to smooth muscle proliferation. Several studies have now shown that agents that promote ROS generation stimulate both systemic and PA smooth muscle proliferation, and suppression of endogenous ROS production inhibits smooth muscle proliferation and promotes apoptosis (6, 7, 32, 47, 60). Moreover, ROS may trigger hypoxia-induced gene transcription by stabilizing hypoxia-inducible factor-1{alpha} (10, 20), a transcription factor known to play a central role in hypoxic pulmonary hypertension (64). The enhanced ROS production, which occurs via NADPH oxidase in our CH IPAs, may contribute to vascular remodeling by augmenting agonist-induced mitogenesis and hypoxia-inducible factor-1-mediated hypoxic gene transcriptions. This is consistent with previous studies that show that suppressing ROS production blunts vascular remodeling and right heart hypertrophy in models of both chronic hypoxic PAH and persistent PAH of the newborn (6, 16, 25). It has been noted that acute hypoxic pulmonary vasoconstriction was unaffected in gp91phox KO mice (2), suggesting that the NADPH oxidase plays differential roles in acute and chronic hypoxic pulmonary hypertension.

NADPH oxidase and enhanced vasoconstrictor responses. Another salient feature of chronic hypoxic PAH is the enhanced pulmonary vascular responsiveness to vasoconstrictor agonists. Several studies have demonstrated that CH enhances pulmonary vasoconstrictor responses to 5-HT in both murine and rat models (28, 31, 38). It has been proposed that this responsiveness is due to increases in the activity of 5-HT1B or 2B receptors (28, 31). Consistent with these previous studies, our data show that CH markedly enhanced murine PA vasoconstriction to 5-HT with about a 60% increase in Emax but only a slight decrease in the EC50. Similar enhanced vasoconstrictor responsiveness was observed for U-46619. These data suggest that CH modulates the activity of both 5-HT and of the thromboxane/prostaglandin pathways. We have previously shown that either exogenously generated superoxide or a KO of extracellular SOD (both conditions that increase superoxide levels) enhance 5-HT-induced pulmonary vasoconstriction. In contrast, conditions that decrease extracellular superoxide levels (e.g., addition of CuZn-SOD or the addition of the NADPH oxidase inhibitor apocynin) reduce 5-HT-induced vasoconstrictor response (35). Based on these findings, we hypothesized that since CH increases extracellular superoxide production, this would lead to enhanced agonist-induced IPA vasoconstriction. This notion is supported by our results that CH-enhanced IPA vasoconstriction to 5-HT and U-46619 was significantly reduced after exogenous application of CuZn-SOD in PAs of WT mice and was similarly blunted in PAs of gp91phox KO mice. Because SOD would not have been expected to rapidly traverse the cellular membrane due to its 32-kDa molecular mass (35–37), it is presumed that superoxide is functionally active in the extracellular milieu. Thus superoxide could be produced in the extracellular space directly or it could be overproduced in the intracellular space after which it is released or diffuses into the extracellular space. However, the mechanism by which superoxide could diffuse across the plasma membrane into the extracellular space is not clear, although others have reported a bicarbonate-chloride anion exchange protein (AE2)-dependent process that actively exchanges intracellular superoxide with extracellular bicarbonate (45). The mechanism for the enhancement of agonist-induced vasoconstriction by superoxide overproduction is also uncertain. One potential mechanism is that superoxide may decrease nitric oxide bioavailability as a result of increased nitric oxide scavenging (44). Because both 5-HT and thromboxane A2 receptors are G protein-coupled receptors (9), it is also possible that superoxide amplifies downstream signaling pathways of these receptors. The potentiation of vasoconstrictor responses could also be mediated, at least in part, by an increase or shift in receptor expression after chronic hypoxia (28). This might be reflected by the reduced, but yet significant, increase in the CH-enhanced IPA constriction to 5-HT and U-46619 in the presence of exogenous CuZn-SOD.

In summary, the present study demonstrates that CH increases superoxide production in IPA via a gp91phox-dependent NADPH oxidase pathway. This increase in superoxide production enhances agonist-mediated IPA vasomotor constrictor activity and remodeling of pulmonary arteriole smooth muscle, both of which contribute to the pathogenesis of PAH. Therefore, the development of therapeutic agents that modulate IPA superoxide production may be an attractive target for the treatment and/or prevention of PAH.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported, in part, by an American Heart Association (Mid-Atlantic Affiliates) Beginning Grant-in-Aid (to J. Q. Liu) and National Heart, Lung, and Blood Institute Grants R01-HL-075134 (to J. S. K. Sham) and R01-HL-64894 (to R. J. Folz).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Q. Liu, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke Univ. Medical Center, MSRB 341, Durham, NC 27710 (e-mail: john.liu{at}duke.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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