Department of Respiratory Medicine and Allergy, The Guy's, King's, and St. Thomas' School of Medicine, King's College London, London SE1 9RT, United Kingdom
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ABSTRACT |
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Increased airway smooth muscle (ASM) content is characteristic of infants with chronic lung disease of prematurity/bronchopulmonary dysplasia. Oxygen therapy, reactive oxygen species (ROS), and immature antioxidant defenses are major risk factors in chronic lung disease of prematurity/bronchopulmonary dysplasia, but their interrelationship is unclear. The direct effects of raised PO2 and modulation of ROS were examined on proliferation of cultured fetal human ASM cells. A bell-shaped relationship was found between PO2 and DNA synthesis induced by fetal bovine serum, platelet-derived growth factor, and basic fibroblastic growth factor, with peak responses occurring at 10-kPa PO2. Changes in DNA synthesis by PO2 did not occur in the absence of mitogen. ROS generation, estimated by dichlorodihydrofluorescein oxidation, was increased by mitogens but was unaffected by nonmitogens (bradykinin, histamine). There was an inverse relationship between ROS generation and PO2, and mitogen-induced ROS generation was substantially potentiated as the PO2 fell. H2O2 mimicked the effect of PO2 on fetal bovine serum-stimulated proliferation, whereas treatment with antioxidants (GSH, N-acetylcysteine) reduced it. These data demonstrate that increases in PO2 above levels found in utero modulate proliferation of fetal ASM cells but only in the presence of growth factors. They also strongly suggest that, under these conditions, proliferation is mediated in part by generation of ROS.
chronic lung disease of prematurity; bronchopulmonary dysplasia; reactive oxygen species; airway remodeling
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INTRODUCTION |
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INFANTS WITH CHRONIC LUNG disease of prematurity/bronchopulmonary dysplasia (CLD-BPD) exhibit airway hyperresponsiveness (AHR), poorly reversible airflow obstruction (27, 46), and, on examination postmortem, the walls of their airways are thickened. This latter feature is associated with airway wall inflammatory processes and increases in the airway wall smooth muscle (ASM) content (5, 22, 45). As in adult humans, structural factors, such as airway wall thickness, cellular and matrix composition, and airway caliber, are key elements likely to limit airway function in infants. James et al. (23) and Lambert et al. (28) have suggested several possible mechanisms by which increased airway wall thickness and increased ASM content, in particular, lead to the development of AHR and airflow obstruction in asthma. By analogy, similar structural changes in abnormally developed airways are likely to also be key elements in development of airflow obstruction in infants with CLD-BPD. In direct support of this, Solway and Hershenson (42) have linked AHR to increased ASM mass in a model of CLD-BPD in immature rats.
Alterations in CLD-BPD airway wall architecture are intimately associated with lung injury and altered repair mechanisms. Major injury-related risk factors in the lungs of infants with CLD-BPD include barotrauma, due to forced mechanical ventilation, prolonged oxygen therapy, free-radical or reactive oxygen species (ROS) generation, immature pulmonary antioxidant defense systems, and release of proteolytic enzymes into the milieu of the airway wall by a predominantly neutrophil-led inflammatory response (2, 31, 48). However, the relationship between lung injury as a result of these risk factors and accelerated ASM growth in infants who develop CLD-BPD is unclear. Evidence from cell culture studies using adult ASM cells suggests that several polypeptide growth factors, proinflammatory cytokines, and other mediators stimulate mitogenesis (see Ref. 19 for review). Of these known ASM mitogens, increased expression of platelet-derived growth factor (PDGF) mRNA and increased production of basic fibroblastic growth factor (bFGF) and insulin-like growth factor-I have been identified in hyperoxic models of CLD-BPD in immature rats (7, 17, 18). Additionally, barotrauma or stretch and increased oxygen or oxidant stress are also potential stimuli that mediate ASM cell proliferation (35, 39, 41) and may modulate cell growth in the developing lung (24, 38).
The relationship among oxygen, oxidant stress, and accelerated cellular
proliferation of fetal human ASM is, however, unknown. Recent studies
have reported that ROS generation paradoxically increases in several
cell types as the PO2 falls (see Ref.
9). In this study, we, therefore, performed experiments to
characterize fetal human tracheal ASM cells in culture and to determine
the direct effects of a range of PO2 (and
procedures designed to modulate oxidant stress) on proliferation of
these cells. In addition, we have examined the relationship among
mitogen stimulation, PO2, and ROS generation in
these cells. Our results demonstrate the first evidence in vitro that
exposure of fetal human ASM to moderate relative hyperoxia (i.e., 10 kPa compared with ~5 kPa in utero) stimulates increased cellular
proliferation, whereas increasing PO2 to 20
kPa causes a progressive decrease in proliferation. This bell-shaped
response to PO2 was dependent on the presence of a mitogenic stimulus. Our results strongly suggest that the relationship between proliferation and PO2 is
dependent on production of ROS, in that it could be mimicked by
application of increasing concentrations of
H2O2 and suppressed by antioxidants and that polypeptide growth factors that stimulate fetal tracheal smooth muscle
cell proliferation also increased ROS generation. Although consistent
with the above, an important additional finding, contrary to the
generally understood relationship, was that the rate of ROS generation
actually increased as PO2 fell, and this
increase in rate was substantially potentiated in the presence of mitogen.
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METHODS |
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Isolation and culture of fetal human trachealis smooth muscle cells. In accordance with procedures approved by the Ethics Committee of St. Thomas' Hospital and The West Lambeth Health Authority, anonymous fetal human tracheae were provided by The Medical Research Council Tissue Bank (Hammersmith Hospital, London, UK) and transported on ice within 6 h of elective termination of pregnancy in RPMI-1640 media containing penicillin-streptomycin (100 U/ml) mixture (Life Technologies, Paisley, UK). Estimated postconceptional age of the tissue ranged from 12 to 18 wk. On arrival, tracheae were transferred into a dissecting tray containing ice-cold Hanks' balanced salt solution (HBSS). The larynx and adventitia surrounding the trachea were cleared, and the epithelium overlying the trachealis muscle was removed by firm scraping of the luminal surface with a rounded scalpel blade. The trachealis was then removed from its cartilage attachments and placed into a 35-mm-diameter dish containing 1.5 ml of HBSS, 10 mg/ml BSA, collagenase (type II, 1 mg/ml), and elastase (type IV, 15 U/ml). The trachealis strip was then chopped finely into small pieces (~1 mm3) and then incubated at 37°C for 40 min.
The above procedure resulted in almost complete disassociation of the tissue into single cells. Initial studies using Trypan blue exclusion showed that at least 90% of the dispersed cells were viable. The cell suspension was centrifuged (200 g for 5 min), and the cell pellet was resuspended in DMEM containing 10% fetal bovine serum (FBS) supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), nonessential amino acid mixture (1×), gentamicin (50 µg/ml), and amphotericin B (1.5 µg/ml). Cells were seeded into a single 12.5-cm2 tissue culture flask. All cultures were initially kept in a CO2-controlled humidified incubator at 37°C with 20-kPa PO2 and 5-kPa PCO2 (normal incubator conditions). Spent culture medium was replaced with fresh every 72 h. At confluence, the cells were successively subcultivated by first scraping into 25 cm2 and then into a 75-cm2 flask. Experiments were performed on cells maintained in culture for up to six passages.Cell acclimatization and stimulation in variable PO2 environments. Before carrying out experiments, cells were first "acclimatized" in a PO2 of 5 kPa, a level approximate to that in the umbilical blood supply in utero (3.3-5.9 kPa) (30). Near-confluent cells, harvested by treatment with 0.01% trypsin-EDTA, were seeded (5 × 104 cells/well) into 24-well plates in DMEM containing 10% FBS. After attachment for 24 h in normal incubator conditions, cells in DMEM containing 0.5% FBS were maintained for 72 h in humidified airtight jars (modified anaerobic "Oxoid" jar, BDH, Poole, UK) that were gassed at 37°C with a O2-5% CO2-balance N2 mixture to provide a PO2 of 5 kPa. Cell growth was initiated by replacement of the 0.5% FBS-containing medium with fresh DMEM containing 0.5, 2.5, or 10% FBS. Cell culture plates were then randomly divided into those placed into humidified airtight jars and gassed as above to provide an experimental PO2 of between 5 and 50 kPa, as appropriate, or those placed directly into the incubator (20-kPa PO2). Jars were regassed every 48 h when spent media were replaced with fresh. Isobaric conditions were maintained in all of the jars by using a one-way bleed valve. PO2 was monitored with an O2 electrode sealed into the jar and did not change significantly from the set value during the period of the experiment. The PO2 and pH of the medium bathing the cells under the above conditions were also examined in five separate experiments to confirm that cells were actually exposed to the appropriate PO2 and normal pH. Cell-conditioned medium was examined by using a blood-gas analyzer (Corning Instruments). In all cases, the pH was ~7.25, and, as expected, the PO2 of the media was not significantly different from that measured by the O2 electrode.
MTT reduction. Proliferation of fetal human ASM cells was determined by mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after removal of cells from jars on days 2, 4, 7, and 9 after initiation of growth. All MTT reduction assays were performed at 20-kPa PO2 to obviate any effects of varying PO2 on the assay itself. Cell monolayers were washed twice in 500-µl DMEM containing 10% FBS. MTT (100 µl of 5 mg/ml in PBS; final concentration 0.8 mg/ml) was then added to each well, and the cells were incubated at 37°C for 6 h before overnight solubilization in a further 500-µl SDS (10% in 0.01 M HCl). A sample (150 µl) from each duplicate well was then transferred to a 96-well microplate, and the optical density was determined by automated dual-wavelength spectrophotometry (Anthos HTII, Salzburg, Austria). The application of this method to human adult ASM cell proliferation has already been described in detail (20). For direct determinations of cell number, cells were detached for counting by hemocytometry by using a trypsin-EDTA solution (0.01% in PBS).
DNA synthesis by using [3H]thymidine labeling. Cells were harvested by using trypsin-EDTA and seeded (5 × 104 cells/well) in 24-well cluster plates in DMEM containing 10% FBS and grown for 7-9 days in normal incubator conditions. Near-confluent cells were then acclimatized as above in DMEM containing 0.5% FBS for 72 h. Thereafter, cell growth was initiated by replacement of the 0.5% FBS with fresh DMEM containing either FBS (0.5, 2.5, or 10%), or 20 ng/ml of recombinant human PDGF isoforms, or bFGF. Each well was also pulsed with [methyl-3H]thymidine (1 µCi/well). Cells were then randomly divided into airtight gas jars in which isobaric atmospheres were maintained with mixtures of 5-kPa CO2 and a range of PO2 (2.5-50 kPa) balanced with N2. After 24 h, stimulation was stopped by removing the radioactive media, washing with ice-cold PBS, and fixing with ice-cold methanol. Cell monolayers were then exposed to ice-cold 5% trichloroacetic acid, and the acid insoluble fraction was lysed in 0.3 M NaOH. Incorporated radioactivity was determined by liquid scintillation spectrometry. Because only three airtight jars were available, ASM cell DNA synthesis studies were limited to assessing the effects of four different oxygen concentrations in any given experiment but always included cells kept at 10- and 20-kPa PO2.
Measurement of intracellular ROS generation. Subconfluent, growth-arrested cells on glass coverslips kept in normal incubator conditions were washed twice in PBS and loaded with 10 µM dichlorodihydrofluorescein diacetate (H2DCF-DA, Molecular Probes, Leiden, The Netherlands) in PBS containing 0.1% BSA for 30 min at 37°C. Cells on coverslips were washed for a further 10 min in H2DCF-DA-free PBS to allow deesterification and then placed in an airtight perfusion chamber (Warner Instrument, Hamden, CT) positioned on a Zeiss Axiovert 200 inverted microscope. Cells were illuminated with an excitation wavelength of 490 nm, and successive images were taken by using a Fluar ×100 oil immersion objective, Sedat Quad dichroic mirror and emitter (Chroma Technology, Brattleboro, VT), and MetaFluor software (Universal Imaging, Downingtown, PA). To minimize photooxidation of H2DCF, the amount of incident light was reduced to the minimum necessary by using neutral density filters. Exposure time was 25-50 ms with 10-20 s between successive images. Changes in PO2 were achieved by fast perfusion (5 ml/min) of the cell chamber with a physiological salt solution at 37°C, containing 118 mM NaCl, 24 mM NaHCO3, 1 mM MgSO4, 4 mM KCl, 5.56 mM glucose, 5 mM sodium pyruvate, 0.435 NaH2PO4, and 1.8 mM CaCl2, pH 7.4, and continuously bubbled with gas mixtures of 5-kPa CO2 and a range of PO2 (2.5-50 kPa) balanced with N2.
Morphological and immunocytochemical characterization of fetal
human tracheal smooth muscle cells.
For localization of smooth muscle contractile proteins, cells were
grown to near confluence on glass coverslips in DMEM containing 10%
FBS and placed into 0.5% FBS for 48 h. Cell monolayers were rinsed twice in ice-cold Ca2+-Mg2+-free PBS and
fixed with ice-cold methanol. Cells were permeablized by using Tween 20 (0.01% in PBS), and nonspecific antibody binding was blocked with 1%
normal goat serum in PBS. Mouse anti-human monoclonal primary
antibodies to smooth muscle -actin (sm-
-actin) (1:500 dilution in
0.1% BSA in PBS) or smooth muscle myosin heavy chain (sm-MHC) (1:500)
were incubated with cells for 1 h at 37°C followed by washing in
PBS by a goat anti-mouse FITC-conjugated secondary antibody (1:100
dilution in 0.1% BSA in PBS; 1 h at 37°C). In control
experiments, primary and/or secondary antibodies were omitted from the
protocol, and cells were incubated in PBS containing
concentration-matched, isotype-matched controls.
Western immunoblot detection of HO-1 expression.
Cells in 25-cm2 flasks, stimulated in exactly the same
fashion as in the immunocytochemical studies, were washed once with ice-cold PBS containing protease inhibitors (200 µM
Na3VO4, 2 mM phenylmethylsulfonyl fluoride) and
then lysed by scraping in ice-cold extraction buffer (20 mM Tris, pH
7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM -glycerolphosphate, 2 mM
Na3VO4, 1 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride). Samples of total protein extracts (10 µg/lane) were separated by SDS-PAGE on 10% acrylamide precast gels
(Invitrogen, Paisley, UK) and transferred electrophoretically to
nitrocellulose membranes. Blots were blocked in PBS containing Tween 20 (0.05%) and dried nonfat milk (5%) for at least 1 h before incubation with an anti-HO-1 polyclonal antibody (1:2,000, catalogue #OSA-100, StressGen Biotechnologies) for 90 min. HO-1 expression was
detected by using a goat anti-mouse IgG horseradish
peroxidase-conjugated secondary antibody (1:5,000, Santa Cruz
Biotechnology) and visualized by enhanced chemiluminescence
(Amersham-Pharmacia, Amersham, UK). To control for small differences in
loading, blots were stripped and reprobed with a mouse anti-
-actin
monoclonal antibody (1:5,000, Sigma Chemical, Poole, UK) and detected
with the goat anti-mouse IgG horseradish peroxidase-conjugated
secondary antibody (1:5,000).
Data analysis. Data in the text and Figs. 2, 3, 5, 6, and 7 legends are expressed as means ± SE of observations obtained from ASM cells cultured from n fetal donors. IC50 values and extrapolated maximum responses were estimated for individual concentration-response curves by using nonlinear least squares regression (SigmaPlot; SPSS, Chicago, IL) where appropriate. IC50 values were converted to negative logarithmic values for all statistical analyses, although, for ease of comprehension, IC50 values are given in the text. Data were compared by using one-way or, when stated, two-way ANOVA followed by a Bonferroni test post hoc (SigmaStat, SPSS). A probability value of <0.05 was considered significant.
Materials.
Recombinant human PDGF isoforms, bFGF, PBS
(Ca2+-Mg2+ free), DMEM, FBS, sodium pyruvate,
L-glutamine, nonessential amino acids, amphotericin B and
gentamicin, and penicillin-streptomycin were obtained from Invitrogen.
EDTA (disodium salt) was obtained from BDH.
[methyl-3H]thymidine was purchased from
Amersham-Pharmacia. H2DCF-DA was purchased from Molecular
Probes. All gases were of research grade (British Oxygen). All other
reagents used in this study, including human reactive, mouse monoclonal
primary antibodies against sm--actin (clone no. 1A4) and sm-MHC
(clone no. h-SMV) and goat anti-mouse FITC-conjugated IgG secondary
antibody, were obtained from Sigma Chemical.
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RESULTS |
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Fetal human ASM cell morphology and contractile protein expression
in culture.
Immunocytochemical localization of smooth muscle contractile proteins
demonstrated that >95% of fetal human tracheal smooth muscle
cells in primary culture expressed sm--actin (Fig.
1A) and sm-MHC (Fig.
1B). Additionally, epithelial cells were not found to be a
contaminating feature of any of the cultures examined because staining
with a monoclonal antibody to pancytokerratin was not detected (data
not shown). At confluence, cultures exhibited a typical
"hill-and-valley" pattern (not shown), which is characteristic of
cultured tracheal smooth muscle cells but not of fibroblasts (19) and is similar to that described for cultured
vascular smooth muscle cells by Campbell and Campbell (8).
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Oxygen modulation of fetal human ASM proliferation is serum
dependent.
FBS-stimulated proliferation of fetal human ASM cell was assessed by
direct hemacytometer counts and by the MTT reduction assay and was both
time and concentration dependent (Fig.
2). While differences in FBS-stimulated
proliferation due to varying PO2 atmospheres
were not present at day 2 of stimulation (P > 0.05 by ANOVA, n = 5), subsequent proliferation
induced by 10% FBS in cells kept in an atmosphere of 10-kPa
PO2 was significantly greater at days
4, 7, and 9 compared with similar cells
maintained in either 5- or 20-kPa PO2, implying
a bell-shaped relationship between proliferation and
PO2 (P < 0.01-0.001,
n = 5, Fig. 2, A and B).
Similarly, at 10-kPa PO2, proliferation induced
by 2.5% FBS was also significantly greater on days 4,
7, and 9 compared with cells maintained at 5- or
20-kPa PO2 (P < 0.05-0.001, n = 5, Fig. 2C). However,
in the presence of 0.5% FBS, which did not itself stimulate
significant proliferation until day 9 (not shown), varying
the PO2 did not induce further proliferation
(P > 0.05 by ANOVA, n = 5, Fig.
2D).
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Oxygen modulates polypeptide growth factor-stimulated DNA synthesis
in fetal human ASM.
To characterize in more detail the apparent bell-shaped relationship
between fetal ASM cell proliferation and PO2
(Fig. 2), it was necessary to examine the effects of a wider range of
isobaric oxygen concentrations (2.5-50 kPa). This necessitated the
use of separate pregassed airtight jars for each
PO2 concentration. Because only three airtight
jars were available, proliferation was, therefore, determined at a
single time point by DNA synthesis-a surrogate marker for cell
proliferation. The existence of a bell-shaped relationship for DNA
synthesis and environmental PO2 was confirmed in cells stimulated with either 10 or 2.5% FBS but was not present in
cells stimulated with 0.5% FBS (Fig.
3A). Isobaric
PO2 levels from 2.5 to 10 kPa caused a
concentration-dependent increase in the sensitivity of fetal ASM cells
to both 2.5 and 10% FBS with maximum DNA synthesis occurring at 10-kPa
PO2 (Fig. 3A). The peak response at
10-kPa PO2 was significantly different
(P < 0.001, n = 4-8) compared
with that induced by either FBS concentration in 20-kPa
PO2 (normal incubator conditions). At 12-kPa
PO2, FBS-stimulated DNA synthesis was less than
at 10 kPa but remained significantly elevated (P < 0.001, n = 4-8) compared with DNA synthesis at
20-kPa PO2. Consistent with data presented in
Fig. 2, PO2 did not affect baseline DNA
synthesis (P > 0.05 by ANOVA, n = 4-8) when FBS was present at only 0.5%.
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Mitogen treatment and reduced PO2 increase
ROS generation.
Production of ROS has been reported on growth factor stimulation of
both vascular (43) and adult bovine tracheal ASM cells (35). To examine whether the mitogens, which stimulated
increased DNA synthesis in fetal human ASM (Fig. 3B), also
induced intracellular generation of ROS, cells were loaded with 10 µM
H2DCF-DA. H2DCF-DA enters the cells and is
deesterified, entrapping nonfluorescent H2DCF inside. The
rate of oxidation of intracellular H2DCF by ROS,
particularly hydrogen peroxide (H2O2;
indirectly via the Fenton reaction and by peroxidases), and by hydroxyl
radicals yields the fluorescent product 2',7'-dichlorofluorescein (DCF) in direct proportion to ROS generation (9, 26). In all
experiments, a steady increase in baseline DCF emission with time was
observed under resting conditions. Application of PDGF-BB
(10 ng/ml) induced a marked increase (~6-10-fold) in the
rate of increase in DCF fluorescence (Fig.
4). Similar increases were observed with
bFGF (20 ng/ml) and 1% FBS (not shown). In contrast, neither 10 µM histamine nor bradykinin (data not shown) had any effect on the rate of
H2DCF oxidation (Fig. 4) and, consistent with reports in
adult human ASM (33), did not stimulate fetal human ASM
proliferation (n = 3 for both, data not shown).
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H2O2 treatment increases FBS-stimulated
proliferation.
To investigate whether exogenous ROS could mimic the effects of
PO2, H2O2, a stronger
oxidant than molecular oxygen, was examined on FBS-stimulated fetal
human ASM proliferation. Similar to findings with varying
PO2 (Figs. 2 and 3), a bell-shaped relationship
for proliferation and increasing H2O2
concentration were observed in cells stimulated for 9 days with 2.5%
FBS (Fig. 6). The maximum extent of
proliferation induced by 2.5% FBS occurred in the presence of 1 µM
H2O2 and was significantly greater
(P < 0.05, n = 4) than that induced by
FBS alone. However, as the concentration of
H2O2 increased >1 µM, proliferation was
progressively inhibited, such that, at 100 µM, proliferation was
significantly below that in the absence of H2O2
(P < 0.05, n = 4; Fig. 6). As would be
expected, in four experiments, exogenous H2O2
also increased the rate of H2DCF oxidation in these cells
(data not shown).
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GSH and N-acetylcysteine inhibit proliferation.
To further determine whether the observed ROS generation was an
epiphenomenon or had a critical role in promoting fetal human ASM
proliferation, the small-molecule, sulfhydryl-containing antioxidants GSH and N-acetylcysteine (NAC) were examined on
FBS-stimulated fetal human ASM proliferation at 10-kPa
PO2, the optimum oxygen environment for
proliferation of these cells (compare Figs. 2 and 3). Treatment with
either GSH or NAC caused a significant decrease in FBS-stimulated
proliferation (Fig. 7). IC50
was 11 ± 2 and 87 ± 16 µM, respectively, with GSH being
around eightfold more potent (P < 0.001, n = 4).
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Increased oxygen levels regulate HO-1 expression in proliferating
cells.
HO-1 expression has previously been used as a marker of oxidant stress
(12, 34). HO-1 expression was, therefore, examined by
Western immunoblot in whole lysates of proliferating and
nonproliferating cells maintained for 24 h at 10-kPa
PO2, when maximal proliferation occurred (Figs.
2 and 3) or at 20-kPa PO2 (normal incubator
conditions). Detectable levels of HO-1 expression were identified in
nonproliferating cells (0.5% FBS) but were found to be unaffected by
PO2. In contrast, HO-1 levels in proliferating
cells (10% FBS) were increased compared with nonproliferating cells,
with the greatest expression occurring in cells cultured at 20-kPa
PO2 (Fig.
8A).
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DISCUSSION |
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This is the first study to report proliferative changes in ASM
cells cultured from fetal human lung tissue. The cells used in our
study fulfilled the accepted morphological and biochemical criteria for
identification of smooth muscle cells in primary culture, including
uniform expression of sm--actin and sm-MHC, as well as development
of a contoured hill-and-valley appearance at confluence (8,
19). Negative pancytokeratin staining indicated that airway
epithelial cells did not contaminate the cultures. The major finding of
this study is that changes in PO2 modulate the
sensitivity of fetal human ASM cells to mitogens, and our results
strongly imply that ROS generation is a key signaling component for
this response. In particular, treatment with the ASM mitogens PDGF-BB,
bFGF, and FBS (but not the nonmitogens bradykinin or histamine)
increased intracellular ROS generation, as estimated by
H2DCF oxidation, and this effect was potentiated at low
PO2. Moreover, exogenous
H2O2, one of the main ROS detected by
H2DCF oxidation, mimicked the effect of oxygen, and
proliferation induced by FBS at 10-kPa PO2 was
inhibited by small-molecule thiol-containing antioxidants (GSH and NAC).
A consequence of preterm birth is a premature marked rise in systemic arterial PO2 above that normally found in utero. The association between changes in the fetal environment brought about by preterm delivery and CLD-BPD has led to suggestions that physicochemical factors could influence ASM cell proliferation in the fetal lung. To test this hypothesis, experiments were designed to investigate the effects of varying oxygen environments and ROS generation on mitogen-stimulated fetal ASM cell DNA synthesis and proliferation. To better reflect the relatively "normoxic" environment in utero for fetal tissues, where the arterial PO2 is much lower (~3.3-5.9 kPa) (30), cultured fetal human ASM cells were initially acclimatized to a baseline PO2 of 5 kPa over 72 h before mitogen stimulation or exposure to higher oxygen-containing environments. Thus exposure of these acclimatized fetal ASM cells to higher PO2 levels was used to model the conditions of relative hyperoxia occurring at birth and with oxygen therapy.
Using this study design, we found that a bell-shaped relationship existed between mitogen-stimulated (FBS; PDGF-AA, -BB, and -AB; and bFGF) ASM cell proliferation and PO2. A twofold increase in baseline PO2 from 5 to 10 kPa enhanced mitogen-stimulated proliferation of fetal ASM cells, and peak proliferation was observed at 10 kPa. In contrast, a twofold reduction in baseline PO2 to 2.5 kPa inhibited mitogen-stimulated fetal human ASM proliferation, and PO2 >20 kPa also inhibited proliferation. Importantly, in the absence of mitogen, no relationship between PO2 and ASM cell proliferation was found, emphasizing a requirement for the presence of mitogen before the modulatory effects of oxygen could be manifest. A similar bell-shaped relationship between PO2 and cell proliferation has been reported in human retinal pigment epithelial cells, with a peak rate of proliferation of ~8-kPa PO2 (4). This and the present study indicate that human cell proliferation is modulated by PO2 within the physiological range. Moreover, the "optimal" PO2 for fetal ASM cell proliferation in vitro is above (i.e., relatively hyperoxic) that found in the fetal environment in utero (~3.3-5.9 kPa) (30). We did not observe differences in cell viability at any of the PO2 levels examined, suggesting that differences in the extent of proliferation at these PO2 were not the result of oxygen-induced cell death. Indeed, studies in adult bovine ASM (39) and in airway epithelial cells (32) have demonstrated that high levels of oxygen are not toxic but induce cell cycle arrest by increased expression of cell cycle-dependent protein kinase inhibitors such as p53 and p21WAF1/CIP1. These effects were associated with exposure to very high oxygen fractions (>50-95%) that are outside the physiological range and exceed 50 kPa, the maximum concentration investigated in the present study. Indeed, it should be noted that not only severe hyperoxia (32, 39) but also hypoxia [~1.5 kPa (10)] can increase expression of p53 and p21WAF1/CIP1.
The mechanism underlying the relationship between PO2 and accelerated mitogen-stimulated fetal ASM cell proliferation is unknown, although there has been considerable interest in the role of ROS, particularly superoxide, H2O2, and hydroxyl radicals (24). Accumulating evidence suggests that ROS are not only injurious by-products of cellular metabolism but, along with the overall oxidant status of a cell, are also essential participants in intracellular signaling and the regulation of mitogenic responsiveness and transcription (6, 9, 12, 37, 43). H2O2 has been shown to be transiently generated during the signal transduction cascade initiated by polypeptide growth factors such as PDGFs and was required for PDGF-induced tyrosine phosphorylation, mitogen-activated protein (MAP) kinase activation, DNA synthesis, and chemotaxis (43). Page et al. (35) reported increased intracellular ROS generation by PDGF in H2DCF-loaded adult bovine ASM cells. Here we report similar findings in fetal human ASM cells, not only with the B-chain homodimer of PDGF, but also with bFGF and FBS. Additionally, we demonstrate that nonmitogens such as bradykinin and histamine do not increase ROS generation, supporting a relationship in these cells between proliferation and ROS generation. This contrasts with other reports from vascular endothelial cells (40, 44) and human keratinocytes (16), in which bradykinin has been implicated in ROS generation.
Although there is increasing acceptance that ROS generation is an important component in mitogen signaling cascades, controversy exists regarding the effect of PO2 on ROS generation. Intuitively, one might expect ROS to be increased in hyperoxia, as the substrate, molecular oxygen, is in greater supply. Indeed, in many tissues and cells exposed to excessive levels of oxygen, there is an oxidant stress response, characterized by nuclear and mitochondrial DNA strand damage, as well as protein oxidation and lipid peroxidation (14, 47). Other features include measures by the cell to limit the oxidant load by increasing the bioavailability or expression of protective antioxidant molecules such as GSH and HO-1 (12, 24, 34). However, there is now considerable evidence that ROS generation by mitochondria and/or plasma membrane-associated NAD(P)H oxidases are increased by hypoxia and form a key component of hypoxic signaling (9, 26). Consistent with this, we have shown that ROS generation, as estimated by H2DCF oxidation, is rapidly increased in human fetal ASM cells as the PO2 falls (Fig. 5). Moreover, there was a synergistic increase in ROS generation when the PO2 was reduced in the presence of the mitogen PDGF-BB.
Unlike ASM proliferation, however, the relationship between ROS generation and PO2 was not bell shaped. This could reflect the possibility either that ROS generation is an epiphenomenona, which is independent of proliferation, or that ROS stimulates proliferation at low concentrations but suppresses it at high concentrations. We, therefore, examined the effect of exogenous H2O2 on mitogen-stimulated proliferation and found that H2O2 had a similar capacity to modulate FBS-stimulated proliferation of fetal human ASM cells, and, in a fashion, that was reminiscent of the bell-shaped relationship observed for PO2 (compare Figs. 3 and 6). This is consistent with our suggestion that a relatively small increase in ROS stimulates proliferation, whereas higher levels cause suppression, and is also consistent with a recent study showing that superoxide stimulates DNA synthesis in rat fetal lung epithelial cells at low concentrations but inhibits it at high concentrations (24). Other investigators have found that H2O2, often in the millimolar range, induces tyrosine phosphorylation and growth factor receptor activation (13, 15). Rao and Berk (36) reported that H2O2 stimulates rat vascular smooth muscle cell mitogenesis, possibly by activating MAP kinase-dependent pathways. Although we did not examine MAP kinase activation in the present study, previous observations by Abe et al. (1) using bovine adult ASM cells have demonstrated that H2O2 stimulates extracellular regulated kinase activation through successive activation of Raf-1 and MAP kinase kinase 1, key signaling intermediates in the activation of MAP kinase-dependent proliferation events by PDGF and other mitogens in ASM cells (reviewed in Ref. 21).
Taken together, our results strongly imply that ROS generation may be a key component of the intracellular signaling pathways that modulate proliferation under these conditions, rather than just a consequence of activation of these pathways. To test this hypothesis further, we examined whether proliferation was inhibited by exogenous small molecule antioxidants. We found that FBS-induced proliferation at 10-kPa PO2, the point at which maximum stimulation occurred, was inhibited by both GSH and NAC, suggesting that an increase in ROS is indeed a key signaling component of the enhanced proliferative response. Brar et al. (6) have reported similar findings with NAC in FBS-stimulated primary cultures of adult rat tracheal smooth muscle and suggested that the signaling pathways involved in mitogen-stimulated ASM proliferation are in part dependent on generation of partially reduced oxygen species generated by NAD(P)H oxidoreductase flavoproteins.
There is, therefore, accumulating evidence that low levels of ROS stimulate cellular proliferation, possibly via activation of MAP kinase-dependent pathways. Our own results also strongly suggest that the regulation of growth by ambient oxygen is mediated through modulation of ROS generation. However, we additionally show that both a decrease in PO2 below 5 kPa, which, in the presence of mitogen, substantially increases ROS generation, and high concentrations of exogenous ROS depress proliferation. Similar data have also recently been presented for the effects of superoxide (24). It is presently unclear what mechanisms may underlie this depression, although relatively high concentrations of superoxide and H2O2 are known to promote apoptosis (25). The bell-shaped relationship between proliferation and PO2 or H2O2 may thus reflect a change in balance between pro-proliferative and proapoptotic processes, although further experiments are required to test this speculation.
The mechanisms underlying the increase in ROS generation during hypoxia and the synergistic potentiation after mitogen stimulation in human fetal ASM cells are presently unclear but could involve either NAD(P)H oxidases or mitochondria. There is presently considerable interest in mitochondrial ROS generation as a key element in hypoxic signaling (9), but our preliminary results using inhibitors of the electron transport chain suggest that, in these cells, mitochondria may only account for a small proportion of ROS under these conditions (unpublished observations). Mitogen-induced rises in intracellular Ca2+ are also unlikely to underlie the synergistic relationship between PO2 and mitogens, as neither bradykinin nor histamine, which caused marked increases in intracellular Ca2+ in these cells (unpublished observations), had any effect on H2DCF oxidation. Further studies are required to determine whether the ROS involved in proliferation originate from mitochondria or an NAD(P)H oxidase and the mechanism underlying the synergistic effect on ROS generation of mitogen stimulation and reduced PO2.
Increased expression of HO-1 has been used as an indicator of oxidant stress (11, 12, 34). HO-1 catalyzes the rate-limiting step in the oxidative conversion of heme to biliverdin, releasing equimolar levels of bilverdin IXa, iron, and carbon monoxide (11). Of the three known isoforms of heme oxygenase, HO-1 is highly inducible by ROS, and accumulating evidence both in vivo and in vitro suggests that HO-1 plays an important adaptive role in providing cellular and tissue protection against such oxidative stress (11, 34). Our data showing that HO-1 expression is increased and locates to the cytosol in mitogen-stimulated human fetal ASM cells are generally consistent with this, as we show that ROS generation is substantially increased in the presence of mitogens. However, in proliferating cells, HO-1 expression appeared to be greater in those maintained at 20 than at 10 kPa (Fig. 8), although the DCF fluorescence data would suggest that the ROS load was greater at 10 kPa than at 20 kPa (Fig. 5). Indeed, hypoxia has been shown to increase HO-1 expression in several tissue types and cell culture model (29). At the present time, we can only speculate as to the reasons for this seeming inconsistency, which may reflect differences between acute changes in redox state (DCF fluorescence) and more prolonged redox changes (HO-1), such as those in proliferating cells. It is also very likely that certain species of ROS and free radicals induced by hyperoxia are not detectable using H2DCF but are capable of causing cell damage and initiating HO-1 expression. Perhaps consistent with this hypothesis, Kazzaz et al. (25) showed that, whereas hyperoxia causes cell necrosis, superoxide and H2O2 result in apoptosis. More detailed studies are required to elucidate this area.
In summary, we have demonstrated that PO2 modulates mitogen-stimulated fetal human ASM cell proliferation, and our results strongly implicate generation of intracellular ROS as a key signaling event in this response. These observations in vitro provide a plausible link among oxygen stress, antioxidant status, and growth factors in altered smooth muscle content in the airways of infants with CLD-BPD. Although previous studies have shown that hyperoxia inhibits proliferation of cultured ASM cells from adult rats and adult humans (3, 39), the present study suggests that the relationship among mitogen-induced fetal human ASM cell proliferation, PO2, and ROS is more complex and depends on the level of relative hyperoxia. Fetal human ASM cell proliferation in vitro was maximal at a PO2 that would be considered supraphysiological for the environment in utero (10 kPa) but close to normal for healthy neonates breathing air. To achieve airway wall geometry consistent with CLD-BPD, the rate of ASM proliferation in neonates receiving assisted ventilation and oxygen therapy may need to be only slightly above the rate of ASM cell proliferation in utero. Our observations could account for such a difference, because developing airways in a growing lung might be expected to be in a milieu of growth factors. Teasing out the complex interrelationship among environmental PO2, polypeptide growth factors, ROS generation, and cellular antioxidants may further aid our understanding of ASM accumulation in CLD-BPD. Furthermore, it is important to note that our data lend weight to recent reservations concerning the injudicious use of antioxidants in neonatal disease, as this could disrupt normal development processes in the fetal lung (24).
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ACKNOWLEDGEMENTS |
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S. J. Hirst was a recipient of a Wellcome Trust Research Career Development Fellowship.
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FOOTNOTES |
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The authors thank the Special Trustees of Guy's and St. Thomas' Hospitals and the Wellcome Trust (051435, 062554).
Address for reprint requests and other correspondence: S. J. Hirst, Dept. of Respiratory Medicine and Allergy, The Guy's, King's and St. Thomas' School of Medicine, Thomas Guy House, Guy's Hospital Campus, London SE1 9RT, UK (E-mail: stuart.hirst{at}kcl.ac.uk).
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.
August 9, 2002;10.1152/ajplung.00268.2001
Received 18 July 2001; accepted in final form 2 August 2002.
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REFERENCES |
---|
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---|
1.
Abe, MK,
Chao TS,
Solway J,
Rosner R,
and
Hershenson MB.
Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease.
Am J Respir Cell Mol Biol
11:
577-585,
1994[Abstract].
2.
Abman, SH,
and
Groothius JR.
Pathophysiology and treatment of bronchopulmonary dysplasia. Current issues.
Pediatr Clin North Am
41:
277-315,
1994[ISI][Medline].
3.
Absher, M,
Makrides W,
Shapiro P,
and
Evans JN.
Hyperoxia inhibits proliferation of rat tracheal smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
267:
L101-L105,
1994
4.
Akeo, K,
Nagasaki K,
Tanaka KY,
Curran SA,
and
Dorey CK.
Comparison of effects of oxygen and antioxidative enzymes on cell growth between pigment epithelial cells and vascular endothelial cells in vitro.
Ophthalmic Res
24:
357-364,
1993[ISI].
5.
Bonikos, DS,
Bensch KG,
Northway WH,
and
Edwards DR.
Bronchopulmonary dysplasia: the pulmonary pathologic sequelae of necrotising bonchiolitis and pulmonary fibrosis.
Hum Pathol
7:
643-666,
1976[ISI][Medline].
6.
Brar, SS,
Kennedy TP,
Whorton AR,
Murphy TM,
Chitano P,
and
Hoidal JR.
Requirement for reactive oxygen species in serum-induced and platelet-derived growth factor-induced growth of airway smooth muscle.
J Biol Chem
274:
20017-20026,
1999
7.
Buch, S,
Han RNN,
Liu J,
Moore A,
Edelson JD,
Freeman RA,
and
Tanswell KA.
Basic fibroblast growth factor and growth factor gene expression in 85% O2.
Am J Physiol Lung Cell Mol Physiol
268:
L455-L464,
1995
8.
Campbell, JH,
and
Campbell GR.
Culture techniques and their applications to studies of vascular smooth muscle.
Clin Sci (Lond)
85:
501-513,
1993[ISI][Medline].
9.
Chandel, NS,
and
Schumacker PT.
Cellular oxygen sensing by mitochondria: old questions, new insight.
J Appl Physiol
88:
1880-1889,
2000
10.
Chandel, NS,
Vander Heiden MG,
Thompson CB,
and
Schumacker PT.
Redox regulation of p53 during hypoxia.
Oncogene
19:
3840-3848,
2000[ISI][Medline].
11.
Choi, AMK,
and
Alam J.
Heme oxygenase-1: function, regulation and implication of a novel stress-inducible protein in oxidant lung injury.
Am J Respir Cell Mol Biol
15:
9-19,
1996[Abstract].
12.
Droge, W.
Free radicals in the physiological control of cell function.
Physiol Rev
82:
47-95,
2002
13.
Gamou, S,
and
Shimizu N.
Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor.
FEBS Lett
357:
161-164,
1995[ISI][Medline].
14.
Gille, JJ,
and
Joejne H.
Cell culture models of oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia.
Mutat Res
275:
405-414,
1992[ISI][Medline].
15.
Goldkorn, T,
Balaban N,
Matsukuma K,
Chea V,
Gould R,
Last J,
Chan C,
and
Chavez C.
EGF receptor phosphorylation and signaling are targeted by H2O2 redox stress.
Am J Respir Cell Mol Biol
19:
786-798,
1998
16.
Goldman, R,
Moshonov S,
and
Zor U.
Generation of reactive oxygen species in a human keratinocyte cell line: role of calcium.
Arch Biochem Biophys
350:
10-18,
1998[ISI][Medline].
17.
Han, RNN,
Buch S,
Freeman RA,
Post M,
and
Tanswell KA.
Platelet-derived growth factor and growth related genes in rat lung. Effect of exposure to 85% O2.
Am J Physiol Lung Cell Mol Physiol
262:
L140-L146,
1992
18.
Han, RNN,
Han VKM,
Buch S,
Freeman BA,
Post M,
and
Tanswell KA.
Insulin-like growth factor-1 and type 1 growth factor receptor in 85% O2-exposed rat lung.
Am J Physiol Lung Cell Mol Physiol
271:
L139-L149,
1996
19.
Hirst, SJ.
Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma.
Eur Respir J
9:
808-820,
1996
20.
Hirst, SJ,
Barnes PJ,
and
Twort CHC
Quantifying proliferation of cultured human and rabbit airway smooth muscle cells in response to serum and platelet-derived growth factor.
Am J Respir Cell Mol Biol
7:
574-581,
1992[Medline].
21.
Hirst, SJ,
Walker TR,
and
Chilvers E.
Phenotypic modulation and mechanisms of proliferation in airway smooth muscle.
Eur Respir J
16:
159-177,
2000
22.
Hislop, AA,
and
Haworth SG.
Airway size and structure in the normal fetal and infant lung and the effect of premature delivery and artificial ventilation.
Am Rev Respir Dis
140:
1717-1726,
1989[ISI][Medline].
23.
James, AL,
Pare PD,
and
Hogg JC.
The mechanics of airway narrowing in asthma.
Am Rev Respir Dis
139:
242-246,
1989[ISI][Medline].
24.
Jankov, RP,
Negus A,
and
Tanswell AK.
Antioxidants as therapy in the newborn: some words of caution.
Pediatr Res
50:
681-687,
2001
25.
Kazzaz, JA,
Xu J,
Palaia TA,
Mantell L,
Fein AM,
and
Horowitz S.
Cellular oxygen toxicity. Oxidant injury without apoptosis.
J Biol Chem
271:
15182-15186,
1996
26.
Killilea, DW,
Hester R,
Balczon R,
Babal P,
and
Gillespie MN.
Free radical production in hypoxic pulmonary artery smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
279:
L408-L412,
2000
27.
Koumbourilis, AC,
Motoyama EK,
Mutich RL,
Mallory GB,
Walcza SA,
and
Fertal K.
Longitudinal follow-up of lung function from childhood to adolescence in prematurely born patients with neonatal chronic lung disease.
Pediatr Pulmonol
21:
28-34,
1996[ISI][Medline].
28.
Lambert, RK,
Wiggs BR,
Kuwano K,
Hogg JC,
and
Pare PD.
The functional significance of increased airway smooth muscle in asthma and COPD.
J Appl Physiol
74:
2771-2781,
1993[Abstract].
29.
Lee, PJ,
Jiang BH,
Chin BY,
Iyer NV,
Alam J,
Semenza GL,
and
Choi AM.
Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.
J Biol Chem
272:
5375-5381,
1997
30.
Myatt, L.
Umbilical-placental and uterine circulations: circulatory changes during gestation.
In: Pediatric and Perinatal Pathophysiology, edited by Gluckman PD,
and Heyman MA.. London: Hodder and Stoughton, 1993, p. 540-546.
31.
Ogden, BE,
Murphy SA,
Saunders GC,
Pathak D,
and
Johnson JD.
Neonatal lung neutrophils and elastase/proteinase inhibitor imbalances.
Am Rev Respir Dis
130:
817-821,
1984[ISI][Medline].
32.
O'Reilly, MA,
Stervsky RJ,
Stripp BR,
and
Finkelstein JN.
Exposure to hypoxia induces p53 expression in mouse lung epithelium.
Am J Respir Cell Mol Biol
18:
43-50,
1998
33.
Orsini, MJ,
Krymskaya VP,
Eszterhas AJ,
Benovic JL,
Panettieri RA,
and
Penn RB.
MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation.
Am J Physiol Lung Cell Mol Physiol
277:
L479-L488,
1999
34.
Otterbein, LE,
and
Choi AM.
Heme oxygenase: colors of defense against cellular stress.
Am J Physiol Lung Cell Mol Physiol
279:
L1029-L1037,
2000
35.
Page, K,
Li J,
Hodge JA,
Liu PT,
Vanden Hoek TL,
Becker LB,
Pestell RG,
Rosner MR,
and
Hershenson MB.
Characterisation of a Rac1 signaling pathway to cyclin D1 expression in airway smooth muscle cells.
J Biol Chem
274:
22065-22071,
1999
36.
Rao, GN,
and
Berk BC.
Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.
Circ Res
70:
593-599,
1992[Abstract].
37.
Rhee, SG.
Redox signaling: hydrogen peroxide as intracellular messenger.
Exp Mol Med
31:
53-59,
1999[ISI][Medline].
38.
Roman, J.
Effects of calcium channel blockade on mammalian lung branching morphogenesis.
Exp Lung Res
21:
489-502,
1995[ISI][Medline].
39.
Shenberger, JS,
and
Dixon PS.
Oxygen-induced S-phase growth arrest and increases p53 and p21WAF1/CIP1 expression in human bronchial smooth muscle cells.
Am J Respir Cell Mol Biol
21:
395-402,
1999
40.
Shimizu, S,
Ishii M,
Yamamoto T,
Kawanishi T,
Momose K,
and
Kuroiwa Y.
Bradykinin induces generation of reactive oxygen species in bovine aortic endothelial cells.
Res Commun Chem Pathol Pharmacol
84:
301-314,
1994[ISI][Medline].
41.
Smith, PG,
Janiga KE,
and
Bruce MC.
Strain increases airway smooth muscle cell proliferation.
Am J Respir Cell Mol Biol
10:
85-90,
1994[Abstract].
42.
Solway, J,
and
Hershenson MB.
Structural and functional abnormalities of the airways of hyperoxia-exposed immature rats.
Chest
107:
89S-93S,
1995[Medline].
43.
Sundaresan, M,
Yu ZX,
Ferrans VJ,
Irani K,
and
Finkel T.
Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.
Science
270:
296-299,
1995[Abstract].
44.
Sundqvist, T.
Bovine aortic endothelial cells release hydrogen peroxide.
J Cell Physiol
148:
152-156,
1991[ISI][Medline].
45.
Sward-Comunelli, SL,
Sherry MM,
Truog WE,
and
Thibeault DW.
Airway muscle in preterm infants: changes during development.
J Pediatr
130:
570-576,
1997[ISI][Medline].
46.
Tepper, RS,
Morgan WJ,
Cota K,
and
Taussig LM.
Expiratory flow limitation in infants with bronchopulmonary dysplasia.
J Pediatr
109:
1040-1046,
1986[ISI][Medline].
47.
Turrens, JF,
Freeman BA,
and
Crapo JD.
Hyperoxia increases H2O2 release by lung mitochondria and microsomes.
Arch Biochem Biophys
217:
411-421,
1982[ISI][Medline].
48.
White, CW,
and
Repine JE.
Pulmonary antioxidant defense mechanisms.
Exp Lung Res
8:
81-96,
1985[ISI][Medline].