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1 Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; and 2 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling. Because hypoxia might promote generation of oxidative stress in vivo, we hypothesized that oxidative stress may play a role in the hypoxia-induced cardiopulmonary changes and examined the effect of treatment with the antioxidant N-acetylcysteine (NAC) in rats. NAC reduced hypoxia-induced cardiopulmonary alterations at 3 wk of hypoxia. Lung phosphatidylcholine hydroperoxide (PCOOH) increased at days 1 and 7 of the hypoxic exposure, and NAC attenuated the increase in lung PCOOH. Lung xanthine oxidase (XO) activity was elevated from day 1 through day 21, especially during the initial 3 days of the hypoxic exposure. The XO inhibitor allopurinol significantly inhibited the hypoxia-induced increase in lung PCOOH and pulmonary hypertension, and allopurinol treatment only for the initial 3 days also reduced the hypoxia-induced right ventricular hypertrophy and pulmonary vascular thickening. These results suggest that oxidative stress produced by activated XO in the induction phase of hypoxic exposure contributes to the development of chronic hypoxic pulmonary hypertension.
N-acetylcysteine; phosphatidylcholine hydroperoxide; xanthine oxidase; allopurinol; pulmonary vascular remodeling
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CHRONIC HYPOXIA CAUSES PULMONARY hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling in humans (13) and in various animal species (25, 26, 28). These pathophysiological aspects are regarded as important factors that may determine the outcome in patients with various respiratory diseases presenting with chronic hypoxemia (6, 11). Hypoxia may generate oxidative stress as suggested by in vitro (2) and in vivo (4) studies. Hypoxic exposure has also been shown to increase production of platelet-activating factor (PAF) in vivo (3, 24). Because reactive oxygen species stimulate the synthesis of PAF by pulmonary arterial endothelium (16), and, because PAF induces the oxidative burst in macrophages (10) and plays a role in the development of pulmonary vascular remodeling induced by hypoxia (22), we hypothesized that oxidative stress might contribute to pulmonary hypertension and vascular remodeling induced by chronic hypoxia.
To test this hypothesis, we first examined the effect of an antioxidant agent, N-acetylcysteine (NAC) (19, 27), on the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial wall thickening in rats after 3 wk of normobaric hypoxic exposure. We then measured lung phosphatidylcholine hydroperoxide (PCOOH) of the hypoxia-exposed rats as a marker of oxidative stress of the lung tissue.
The xanthine oxidase (XO)-hypoxanthine system has been known to be one of the important pathways to generate oxidative stress in vivo (18), and XO, the final enzyme of purine catabolism, can be activated by hypoxia in cultured pulmonary arterial endothelial cells (23). However, it is not known whether the lung XO activity undergoes a change during hypoxic exposure in vivo and whether XO plays a role in the hypoxia-induced cardiopulmonary changes. We investigated the changes in lung XO activity of hypoxia-exposed rats and the effect of the XO inhibitor allopurinol on lung PCOOH levels and hypoxic pulmonary hypertension.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and hypoxic exposure. Male Sprague-Dawley rats (Funabashi Farm, Sendai, Japan) weighing 175-335 g were used in this study. Rats were exposed to hypoxia in a normobaric hypoxic chamber (60 × 70 × 50 cm). The hypoxic environment was maintained by 5.5 l/min flow of hypoxic air (10% oxygen). This low-oxygen-containing air was produced by a hypoxic gas generator (Teijin, Sendai, Japan) utilizing exhaust air from an absorption-type oxygen concentrator (TO-90, Teijin, Tokyo, Japan). The chamber was opened twice a day for 10 min for feeding. After the chamber was closed, 100% nitrogen gas was flushed into the chamber to obtain the hypoxic environment as quickly as possible. The oxygen concentration in the chamber was measured collecting the gas from the tubing penetrating the chamber wall using a blood gas analyzer (ABL300, Radiometer, Copenhagen, Denmark) before opening it and after flushing with nitrogen gas. Soda lime was placed in the chamber to reduce the concentration of carbon dioxide. Control rats were maintained in room air.
Effects of NAC treatment on pulmonary hypertension, right ventricular hypertrophy, and pulmonary artery media wall thickness induced by chronic hypoxia. Twenty-four rats were divided into two groups: exposed to normoxia (n = 12) or hypoxia (n = 12). Each group was again divided into two subgroups for treatment with water (normoxia + water, n = 6; hypoxia + water, n = 6) or NAC (Senju Pharmaceutical, Osaka, Japan) (normoxia + NAC, n = 6; hypoxia + NAC, n = 6). The rats received water or 1% NAC solution ad libitum from 24 h before the initiation of normoxic or hypoxic exposure. Then they were housed for 3 wk under normoxic conditions or in normobaric hypoxia (10% oxygen). Water or NAC solution was exchanged once a day for 3 wk.
At 3 wk after normoxic or hypoxic exposure, the rats were anesthetized with ketamine hydrochloride (60 mg/kg) and xylazine (8 mg/kg) intramuscularly. The pulmonary arterial pressure was measured after a polyethylene catheter (PE-10, Intramedic, Clay Adams, Parsippany, NJ) had been inserted into the main pulmonary artery through the right jugular vein. The placement of the catheter was guided by the shape of the pressure tracing displayed on an oscilloscope (Polygraph System, Nihon Kohden, Tokyo, Japan). The pressure was measured using a pressure transducer (TP-400T, Nihon Kohden); the zero reference point was the level of the midchest. Afterward, the rats were anesthetized with pentobarbital sodium (100 mg/kg ip) and ventilated with room air via a tracheal cannula (15-gauge Luer stub adapter, Clay Adams) by use of a small animal respirator (Model SN-480-7, Shinano, Tokyo, Japan) at 55 breaths/min with 8 cmH2O inspiratory pressure and 3 cmH2O positive end-expiratory pressure. Next, a midsternotomy was performed, and 100 units of heparin were injected into the right ventricle. After a blood sample was collected by cardiac puncture for measurement of hematocrit, the pulmonary artery was cannulated through an incision in the right ventricle and perfused with Earle's balanced salt solution (37°C, 20 cmH2O pressure) to wash out residual blood, allowing the fluid to drain through an incision in the left ventricle. Then the heart and lungs were removed en bloc, a hot barium gelatin mixture (60°C) was injected at 37.5 mmHg pressure for 2 min into the pulmonary artery, and the airways were distended with 10% formaldehyde solution at 15 cmH2O and fixed in an inflated state in 10% formaldehyde solution for 3 days (25). For the assessment of right ventricular hypertrophy, the hearts were removed and fixed in 10% buffered formaldehyde solution for a minimum of 3 days. The atria, valves, and great vessels were dissected from the ventricles, weights of the right ventricular free wall (RV) and the left ventricle together with the septum (LV + S) were measured separately, and the ratio RV/(LV + S) was calculated. For morphometric analysis, one sagittal section was obtained from each left lung. The section was stained with Elastica-Masson's stain and microscopically assessed for arterial media wall thickness. Measurements of external diameter and media wall thickness were made on at least 30 muscular arteries (in the size range of 50-100, 100-150, and 150-200 µm in external diameter) per lung section at ×400 magnification. For each artery, the media wall thickness (%WT) was expressed as %WT = (media thickness × 2)/external diameter × 100.Measurement of lung PCOOH levels. For the assessment of oxidative stress in the lung tissue, PCOOH levels were measured by using a modification of the chemiluminescence-high-performance liquid chromatography (CL-HPLC) method of Miyazawa et al. (20, 21) described below.
Thirty-eight rats were divided into eight groups. Twenty-four animals were exposed to hypoxia [inspired oxygen fraction (FIO2) = 0.1]. At 1 (n = 6), 3 (n = 6), 7 (n = 4), 14 (n = 4), and 21 (n = 4) days after the initiation of hypoxic exposure, the rats were killed and their lungs were collected as described below. Fourteen rats were maintained in room air (normoxia), and their lungs were collected on days 1 (n = 6), 7 (n = 4), and 21 (n = 4) of the study. At the day of lung sampling, the rats were anesthetized with pentobarbital sodium (100 mg/kg ip), the chest was opened via a median sternotomy, and the lungs were removed en bloc and quickly frozen in liquid nitrogen. All frozen tissues were stored at
80°C until
extraction of total lipid.
Total lipid was extracted from the rat lung by a modification of the
method of Miyazawa et al. (20, 21), using a mixture of
chloroform and methanol (2:1 vol/vol). Two milliliters of 0.15 M NaCl
containing 0.002% butylated hydroxytoluene (BHT) were added as an
antioxidant to 500 mg of lung tissue, and the mixture was homogenized
in a glass-glass homogenizer (7700 Homogi, Pyrex, Iwaki Glass,
Funabashi, Japan). The homogenate was added to 4.5 ml of
chloroform-methanol (2:1 vol/vol) containing 0.002% BHT and was mixed
vigorously for 1 min. The mixture was centrifuged at 3,000 rpm for 10 min at 4°C. The lower chloroform layer containing lung total lipid
was collected. The extraction was repeated three times. After
dehydration with anhydrous sodium sulfate, the chloroform layer was
concentrated in a rotary evaporator and dried under a nitrogen stream.
The lung total lipid obtained was diluted with 100 µl of
chloroform-methanol (2:1 vol/vol), and a 30-µl portion was subjected
to the CL-HPLC assay. The CL-HPLC system consists of normal-phase HPLC
and a hydroperoxide-specific chemiluminescence detector. The HPLC
column (JASCO Fine pak SIL NH2-5) was placed in a column oven
(860-CO, JASCO, Tokyo, Japan; 40°C). The column mobile phase was
isopropanol-methanol-distilled water (26:9:5, vol/vol/vol) and the flow
rate was 1.1 ml/min using a JASCO 880-PU pump. The column eluate was
mixed with a chemiluminescence reagent at a postcolumn mixing joint.
The chemiluminescence reagent was prepared by dissolving 7 µg/ml of
cytochrome c (from horse heart, type VI, Sigma Chemical, St.
Louis, MO) and 2 µg/ml of luminol (3-aminophthalhydrazide,
Wako Pure Chemical, Tokyo, Japan) in 50 mM borate buffer (pH 10.0). The
flow rate of the chemiluminescence reagent was 1.0 ml/min (880-PU pump,
JASCO). The generated chemiluminescence was measured by a
chemiluminescence detector (CLD-100, Tohoku Electronic, Sendai, Japan)
and quantified by an integrator (Sic Chromatocoader 12, JASCO). A
calibration curve was prepared by using authentic PCOOH produced by
photooxidation of egg yolk phosphatidylcholine that had
previously been purified. The concentration of hydroperoxide was
expressed as picomoles of hydroperoxide-oxygen.
Effects of NAC treatment on lung PCOOH levels. Thirty-three rats were divided into seven groups. The normoxia group was kept under normoxic conditions (n = 6). Three groups received water, and the other three groups received 1% NAC solution ad libitum. Twenty-four hours after the initiation of the treatment, the rats in the six treatment groups were kept in normobaric hypoxia (FIO2 = 0.1) with water or NAC continued ad libitum. At 1 (water, n = 6; NAC, n = 6), 7 (water, n = 4; NAC, n = 3), and 21 days (water, n = 4; NAC, n = 4) of hypoxic exposure, lungs were isolated and subjected to the measurement of PCOOH levels as described in Measurement of lung PCOOH levels.
Assay of activities of XO and xanthine dehydrogenase in the lung tissues. XO and xanthine dehydrogenase (XD) activities were assayed by the fluorometric method of Akaike et al. (1) described below.
Twenty-four rats were divided into six groups. Eighteen animals were exposed to hypoxia (FIO2 = 0.1). At 1 (n = 6), 3 (n = 3), 7 (n = 3), 14 (n = 3), and 21 (n = 3) days after the initiation of hypoxic exposure, they were killed and their lungs were collected as described below. Six rats were maintained in room air, and their lungs were collected (normoxia). At the day of lung sampling, the rats were anesthetized with pentobarbital sodium (100 mg/kg ip) and ventilated with room air via a tracheal cannula (15 G Luer stub adapter, Clay Adams) by using a small animal respirator (model SN-480-7, Shinano) at 55 breaths/min with 8 cmH2O inspiratory pressure and 3 cmH2O positive end-expiratory pressure. Next, a midsternotomy was performed, 100 units of heparin were injected into the right ventricle, and the pulmonary artery was cannulated through an incision in the right ventricle and perfused with 0.01 M phosphate-buffered 0.15 M NaCl (PBS; pH 7.4, 37°C, 20 cmH2O pressure), containing 2 mM p-amidinophenylmethanesulfonyl fluoride ·HCl (APMSF; Wako Pure Chemical) and 10 mM dithiothreitol (DTT; Wako Pure Chemical) to wash out residual blood, allowing the fluid to drain through an incision in the left ventricle. Then the left lung was removed, quickly frozen in liquid nitrogen, and stored at80°C until sample treatment.
The left lung was homogenized (Polytron homogenizer; Kinematica GmbH,
Lucerne, Switzerland) with four volumes of ice-cold 50 mM sodium
phosphate buffer pH 7.6 containing 0.1 mM EDTA (Wako Pure Chemical), 2 mM APMSF, 1 mM DTT, 10 µg/ml leupeptin (Wako Pure Chemical), 10 µg/ml trypsin inhibitor (Type 1-S from soybean, Sigma Chemical) and
0.32 M sucrose (Wako Pure Chemical). The homogenates were centrifuged
at 10,000 g for 30 min at 4°C, and the supernatants were dialyzed for
18 h against 5 liters of PBS at 4°C to remove the low
molecular-weight compounds before determination of XO enzyme activity.
Samples were assayed for their XO activity using pterin (Sigma
Chemical) as the substrate in a spectrofluorometer (model 650-40; Hitachi, Tokyo, Japan) with excitation at 345 nm and emission at 390 nm. The volume of the assay mixture was 1.0 ml in 50 mM sodium
phosphate buffer pH 7.6, which consisted of 10 µM pterin and the
putative enzyme from the samples. Reactions proceeded for 10-60
min at 37°C. To measure both XO and XD activity, the above reaction
was carried out in the presence of 10 µM methylene blue. To confirm
the specificity of the activity, 10 µM allopurinol was added to the
above mixture and the reaction was carried out. The concentration of
isoxanthopterin formed was calculated from the linear relationship
between fluorescence intensity vs. its concentration in 0-10 nmol
of the product.
Effects of allopurinol treatment on lung PCOOH levels. Twenty-six rats were divided into five groups. The normoxia group was kept under normoxic conditions (n = 6). Two groups received water, and the other two groups received allopurinol (50 mg/kg) via gavage every 12 h. Two hours after the second treatment, the rats in the four treatment groups were kept in normobaric hypoxia (FIO2 = 0.1). Water or allopurinol was continued as described above. At 1 (water, n = 6; allopurinol, n = 6) and 7 days (water, n = 4; allopurinol, n = 4) of hypoxic exposure, lungs were removed and subjected to measurement of PCOOH levels as described in Measurement of lung PCOOH levels.
Effects of allopurinol treatment on pulmonary hypertension, right ventricular hypertrophy, and pulmonary artery media wall thickening induced by chronic hypoxia. Twenty-five rats were divided into two groups: housed in normoxia (n = 12) or exposed to hypoxia (n = 13). Each group was again divided into two subgroups for treatment with water (normoxia, n = 6; hypoxia, n = 7) or allopurinol (50 mg/kg; normoxia, n = 6; hypoxia, n = 6) via gavage every 12 h. Two hours after the second treatment, they were housed for 3 wk under normoxic conditions or in normobaric hypoxia (10% oxygen) with water or allopurinol continued as described above.
At 3 wk after normoxic or hypoxic exposure, mean pulmonary arterial pressure, RV/(LV + S), hematocrit, and pulmonary arterial media wall thickness were measured as described in Effects of NAC treatment on pulmonary hypertension, right ventricular hypertrophy, and pulmonary artery media wall thickness induced by chronic hypoxia.Effects of allopurinol treatment during the initial 3 days of hypoxic exposure on right ventricular hypertrophy and pulmonary artery media thickening. Twenty-four rats were divided into four groups as in the previous allopurinol study. The rats received water or allopurinol (50 mg/kg) via gavage every 12 h. Two hours after the second treatment, they were housed for 3 wk under normoxic conditions or in normobaric hypoxia (10% oxygen) with water or allopurinol administration continued as described above only during the initial 3 days.
At 3 wk after normoxic or hypoxic exposure, RV/(LV + S) and pulmonary arterial media wall thickness were measured as described in Effects of NAC treatment on pulmonary hypertension, right ventricular hypertrophy, and pulmonary artery media wall thickness induced by chronic hypoxia.Statistical analysis. Values are expressed as means ± SE. Means of several groups were compared by one-way analysis of variance, and those of two groups were compared by unpaired t-test. Differences were considered significant when P < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of NAC treatment on pulmonary hypertension, right
ventricular hypertrophy, and pulmonary artery media thickness induced
by chronic hypoxia.
Three weeks of hypoxic exposure caused pulmonary hypertension, right
ventricular hypertrophy, and polycythemia in rats. Chronic treatment
with NAC significantly reduced pulmonary hypertension and right
ventricular hypertrophy induced by chronic hypoxia but had no effect on
the hematocrit in normoxic or chronically hypoxic rats. Mean pulmonary
arterial pressure and RV/(LV + S) in the NAC-treated normoxic rats
were not different from those in the water-treated normoxic rats (Table
1).
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Measurement of lung PCOOH levels.
The lung PCOOH levels in the hypoxia-exposed rats were higher than
normoxia controls at days 1 and 7. It seems that
the lung PCOOH levels began to increase at 1 day after the initiation
of hypoxic exposure, reached a maximum at 7 days, and then tended to
decline. Three-week aging of animals in the normoxic environment did
not affect the lung PCOOH levels (Fig.
2).
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Effects of NAC treatment on lung PCOOH levels.
At 1 day after the initiation of hypoxic exposure, the lung PCOOH
levels of NAC-treated hypoxic rats were significantly lower than those
of water-treated hypoxic rats (hypoxia + NAC, 137.5 ± 16.7 pmol/g; hypoxia + water, 216.2 ± 18.3 pmol/g). Similarly, at
day 7 of hypoxic exposure, NAC treatment reduced the
increase in the lung PCOOH levels caused by hypoxia (hypoxia + NAC, 124.6 ± 9.5 pmol/g; hypoxia + water, 252.7 ± 96.0 pmol/g). At day 21 of hypoxic exposure, PCOOH levels in the
NAC-treated rat lungs tended to be lower than those in the
water-treated rat lungs (hypoxia + NAC, 123.4 ± 6.2 pmol/g;
hypoxia + water, 231.2 ± 65.4 pmol/g) (Fig.
3).
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Assay of activities of XO and XD in the lung tissues.
XO activities in the hypoxia-exposed rat lungs were elevated at
day 1 of hypoxic exposure (0.458 ± 0.024 nmol
isoxanthopterin · min
1 · ml1
of lung homogenate), reached a maximum at day 3 (0.532 ± 0.049), and were maintained at higher levels compared with those in
the normoxic rat lungs (0.339 ± 0.019) from the 7th to the 21st
day (hypoxia 7 days, 0.433 ± 0.032; 14 days, 0.459 ± 0.006;
21 days, 0.480 ± 0.045) (Fig.
4A). XD activities in the lung
tissue began to increase at 3 days after the initiation of hypoxic
exposure (normoxia, 1.111 ± 0.051; hypoxia 3 days, 1.368 ± 0.025 nmol
isoxanthopterin · min1 · ml1
of lung homogenate) and then tended to increase more gradually until
day 21 (Hypoxia 7 days, 1.418 ± 0.017; 14 days,
1.533 ± 0.089; 21 days, 1.662 ± 0.050) (Fig.
4B). The time course of XO + XD total activities in the
lung tissue was similar to that of XD activities (Fig. 4C).
The XO-to-XD ratio increased only at day 1 of the hypoxic
exposure (normoxia, 0.309 ± 0.022; hypoxia 1 day, 0.401 ± 0.034) (Fig. 4D).
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Effects of allopurinol treatment on lung PCOOH levels.
At days 1 and 7 of hypoxic exposure, treatment
with allopurinol reduced the PCOOH levels in the hypoxia-exposed rat
lungs (hypoxia 1 day + water vs. hypoxia 1 day + allopurinol:
226.9 ± 20.4 vs. 145.9 ± 7.6 pmol/g, P < 0.05; hypoxia 7 days + water vs. hypoxia 7 days + allopurinol: 293.6 ± 52.1 vs. 134.3 ± 6.1 pmol/g,
P < 0.05) (Fig. 5).
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Effects of allopurinol treatment on pulmonary hypertension, right
ventricular hypertrophy, and pulmonary artery media wall thickness.
Three-week hypoxic exposure also caused pulmonary hypertension, right
ventricular hypertrophy, and polycythemia when the rats received water
via gavage twice a day. However, oral administration of allopurinol
significantly decreased hypoxia-induced pulmonary hypertension and
right ventricular hypertrophy, whereas allopurinol itself had no effect
on these parameters in the normoxic rats. Treatment with allopurinol
did not affect the hematocrit in normoxic and chronically hypoxic rats
(Table 3).
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Effects of allopurinol treatment during the initial 3 days of
hypoxic exposure on hypoxia-induced right ventricular hypertrophy and
pulmonary artery media thickening.
Allopurinol treatment during the first 3 days also significantly
inhibited hypoxia-induced right ventricular hypertrophy (normoxia + water, 0.23 ± 0.01; normoxia + allopurinol, 0.24 ± 0.01; hypoxia + water, 0.41 ± 0.01; hypoxia + allopurinol, 0.33 ± 0.01) (Fig. 6A) and media wall thickening
of the pulmonary arterioles in the size range of 50-200 µm
external diameter (Fig. 6B).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The principal findings of our study are as follows: 1) the antioxidant NAC reduced pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular media thickening caused by 3 wk of normobaric hypoxic exposure; 2) lung tissue levels of PCOOH, a primary peroxidation product of phosphatidylcholine, increased from day 1 to day 7 of hypoxic exposure (and reached a maximum at day 7); 3) treatment with NAC inhibited the increase in PCOOH levels in the lungs from the hypoxia-exposed rats; 4) XO activity in the rat lungs was elevated from day 1 through day 21 of the hypoxic exposure (the maximal values occurred at the third day); 5) treatment with a XO inhibitor, allopurinol, reduced the increase in PCOOH levels in hypoxia-exposed rat lungs and attenuated pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular media thickening in the rats exposed to hypoxia for 3 wk; and 6) allopurinol treatment only for the first 3 days of a 3-wk hypoxic exposure period inhibited hypoxia-induced right ventricular hypertrophy and pulmonary vascular media thickening. Taken together, these findings suggest that generation of oxidative stress contributes to the development of pulmonary hypertension and pulmonary vascular thickening induced by chronic hypoxia and that lung XO activation during the early phase of chronic hypoxic exposure is involved in the production of reactive oxygen species.
Hypoxia can cause an increase in rat plasma glutathione disulfide level in vivo (4) and increased formation of lipid peroxidative products in cultured bovine pulmonary endothelial cells in vitro (2), suggesting that hypoxic exposure may promote the generation of oxidative stress in vivo. Hypoxic exposure has also been shown to increase the production of PAF in plasma (3) or in bronchoalveolar lavage fluid (24), and reactive oxygen species stimulate the synthesis of PAF by bovine pulmonary arterial endothelium (16). PAF induces the oxidative burst in macrophages (10) and plays a role in the development of pulmonary vascular remodeling induced by hypoxia (22). It is of interest that hyperoxia induces generation of intracellular free radicals (7) and causes pulmonary artery remodeling and pulmonary hypertension (14, 15). On the basis of these observations, we hypothesized that oxidative stress might contribute to hypoxia-induced pulmonary hypertension and vascular remodeling.
We found that chronic administration of NAC reduced the hypoxia-induced cardiopulmonary alterations and that treatment with NAC had no effect on the hypoxia-related hemoconcentration, which suggests that the reduction of pulmonary hypertension by NAC was not due simply to an alteration of blood viscosity.
The effect of NAC administration on pulmonary artery pressure was less than that on pulmonary arterial media thickening. The reason for that is unclear. The pulmonary vascular remodeling associated with development of pulmonary hypertension is histologically characterized not only by increased wall thickness of the muscular pulmonary arteries but also by abnormal extension of muscle into peripheral arteries, where it is not normally present, and reduction in the arterial tissue density (25). We speculate that one reason for the smaller effect of NAC treatment on the development of pulmonary hypertension could be a smaller effect of NAC on muscularization of nonmuscular peripheral arteries.
Because the treatment with NAC was effective, we hypothesized that hypoxic exposure might cause generation of oxidative stress in the lung tissue. Indeed, when we measured PCOOH in the hypoxia-exposed rat lungs using the CL-HPLC assay (20, 21), we found that the lung PCOOH levels increased from the first day through the seventh day of hypoxic exposure. This indicates that hypoxia induces oxidative stress in the lung tissue during early hypoxic exposure.
The lung PCOOH levels of NAC-treated hypoxic rats were significantly lower than those of water-treated hypoxic rats on day 1, and they were maintained at a low level at the 7th and 21st days of the hypoxic exposure. The fact that NAC reduced both the hypoxia-induced increase in PCOOH levels and the development of pulmonary hypertension indicates that the generation of oxidative stress may contribute to the hypoxia-induced pulmonary hypertension.
In this study, we also measured lung tissue XO, which is the final purine catabolizing enzyme contributing to oxidative stress in vivo (8, 18), by catalyzing the oxidation of hypoxanthine to xanthine and xanthine to uric acid and generating the superoxide anion (18). Superoxide anion is metabolized to H2O2 by disproportionation (17) and promotes lipid peroxidation in the presence of transitional metals (9). Elevation of XO activity has been reported in bovine pulmonary artery endothelial cells exposed to hypoxia (23). Cultures of bovine pulmonary artery endothelial cells accumulate a significant amount of hypoxanthine in their medium, a substrate for XO, when exposed to hypoxia (12). Thus XO activation in the hypoxia-exposed lung might generate oxidative stress. We found indeed that lung tissue XO activity increased at 1 day after the initiation of the hypoxic exposure, then reached a maximum at 3 days, and then tended to decline.
Because XD can be reversibly converted to XO through oxidation of sulfhydryl groups or irreversibly through proteolysis (5), we measured both XD and XO activities.
The lung XD activity and the combined XO plus XD activities of hypoxic rats were significantly higher than those of normoxic rats at third day of the hypoxic exposure and tended to increase gradually from 3rd to 21st day. However, the XO-to-XD ratio increased only at day 1 of the hypoxic exposure, suggesting that hypoxia activates XO in the lung tissue by inducing the conversion of XD to XO early during hypoxia. Subsequently, total XO and XD activities are increased.
Because hypoxic exposure caused lung XO activation, we investigated the effect of allopurinol, a competitive inhibitor of XO, on the generation of oxidative stress and the development of cardiopulmonary changes. Allopurinol significantly inhibited both the increase in lung PCOOH levels and the cardiopulmonary alterations induced by chronic hypoxic exposure. This indicates that XO under hypoxic conditions generates oxidative stress, leading to the development of pulmonary vascular thickening and hypertension, and further that there are apparently two phases during chronic hypoxia. The first induction phase, up to day 7, is characterized by XO activation and generation of oxidative stress, whereas the second phase may be an adaptation phase with further pulmonary vascular remodeling. We hypothesized that inhibition or decrease of the oxidative stress during the first induction phase might result in reduction of the cardiovascular alterations occurring in the subsequent adaptation phase. Indeed, the inhibition of XO only during the initial 3 days of the hypoxic exposure was sufficient to reduce the degree of right ventricular hypertrophy and pulmonary vascular thickening that usually develops during 3 wk of chronic hypoxic exposure. Our interpretation of these findings is that the oxidative stress generated via XO activation during the induction phase of chronic hypoxia plays a role in the development of pulmonary hypertension.
Our data do not address the cell sources of hypoxia-related oxidative stress and the sites of XO activation, but we speculate that pulmonary arterial endothelial cell XO may contribute to the generation of oxidative stress in the hypoxia-exposed rat lungs, because hypoxia causes accumulation of lipid peroxidation products (2) and activation of XO (23) in cultured pulmonary arterial endothelial cells.
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ACKNOWLEDGEMENTS |
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We thank N. Yamaguchi (Teijin, Tokyo, Japan) for constructing the hypoxic gas generator, T. Tsuboi (Senju Pharmaceutical, Osaka, Japan) for the generous gift of NAC, Dr. T. Miyazawa and Dr. M. Kinoshita, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai, Japan for instruction in the CL-HPLC method, Dr. T. Akaike and Dr. K. Umezawa, Department of Microbiology, Kumamoto University School of Medicine, Kumamoto, Japan, for instruction in the fluorometric method to assay xanthine oxidase activities, and Dr. Mark W. Geraci, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, CO, for helpful discussion.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Hoshikawa, Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan (E-mail: yasuhoshi{at}aol.com).
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.
Received 9 November 2000; accepted in final form 22 November 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, Normoxia-housed rats (normoxia 1 day, 7 days, and 21 days); 
, hypoxia-exposed rats (hypoxia 1 day, 3 days, 7 days, 14 days, and 21 days). Number of rats is indicated in
parentheses. *P < 0.05 vs. normoxia 1-day values;
P < 0.05 vs. normoxia 7-day values. Lung PCOOH
levels of the hypoxia-exposed rats were higher than those of normoxia
controls at days 1 and 7 of hypoxic exposure,
whereas aging did not affect the PCOOH levels in the normoxic rat
lungs.
