Hyperoxia inhibits fetal rat lung fibroblast proliferation and
expression of procollagens
Naveed
Hussain,
Fengying
Wu,
Constance
Christian, and
Mitchell J.
Kresch
Division of Neonatology, Department of Pediatrics, University of
Connecticut School of Medicine, Farmington, Connecticut 06030-2203
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ABSTRACT |
The direct effects
of hyperoxia on collagen production by fetal lung fibroblasts are
unknown and would be important to the understanding of the molecular
mechanisms involved in bronchopulmonary dysplasia in premature infants.
We studied the effect of hyperoxia on
1) proliferation,
2) mRNA levels for type I and III
procollagens, and 3) net collagen
production in primary cultures of fetal rat lung fibroblasts.
Fibroblasts from 19-day-old rat fetuses (term is 22 days) were
obtained. Test plates were incubated in hyperoxia and controls in room
air for varying time periods. Cell viability in both conditions was
>97% as determined by trypan blue exclusion. Fibroblast
proliferation in nonconfluent cultures was found to be significantly
reduced with exposure to hyperoxia (P < 0.001). Steady-state levels of type I and III procollagen mRNAs,
analyzed on Northern blots hybridized to
[32P]cDNA probes, were
significantly decreased in hyperoxia
(P < 0.01). This effect was noted as
early as 4 h of exposure to hyperoxia and persisted for 5 days. There
was a significant inverse correlation between duration of exposure to
O2 and steady-state levels of mRNA
for
1(I)-procollagen
(r =
0.904) and
1(III)-procollagen (r =
0.971). There were no
significant changes in steady-state levels of
-actin mRNA. We also
found a significant decrease in collagen synthesis in hyperoxia-exposed
fibroblasts (P < 0.05). We conclude
that hyperoxia directly effects a reduction in fetal lung fibroblast
proliferation and net collagen production at a pretranslational level.
lung fibrosis; type I procollagen; type III procollagen; hyperoxia
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INTRODUCTION |
EXPOSURE OF THE LUNGS of human premature infants to
high concentrations of O2 is a
major factor in the development of bronchopulmonary dysplasia, which is
characterized by inflammation and fibrosis (5). The major contributors
to fibrosis in this situation are collagen types I and III produced by
lung fibroblasts (12). Similar changes are observed in the
O2-exposed lungs of numerous species, including the rat, as reviewed by Balentine (3). The mechanism
by which hyperoxia ultimately leads to lung fibrosis is unclear.
Understanding whether this is a direct effect of hyperoxia on
fibroblasts or secondary to the influence of
O2 on other cell types is
important. The little that is known suggests that hyperoxia inhibits
proliferation of cultured fibroblasts derived from adult animals (29)
or cell lines (4), but it is not known if fetal lung fibroblasts have
the same response. Also, the direct effect of hyperoxia on lung
fibroblast collagen expression is unknown. We formulated the hypothesis
that O2 directly affects
expression of types I and III collagen by fibroblasts derived from
fetal rat lungs. In this report, we describe the direct effects of
hyperoxia on 1) fetal rat lung
fibroblast proliferation, 2)
steady-state levels of mRNA for
1-chains of type I and type III
procollagens, and 3) net collagen
production and noncollagen protein synthesis by fetal rat lung
fibroblasts.
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METHODS AND MATERIALS |
Isolation and culture of fetal lung fibroblasts.
Primary cultures of fibroblasts from fetal rat lungs were obtained as
previously described by Smith (27) and Kresch et al. (22). Briefly, the
lungs of 19-day gestation fetal rats (term is 22 days) were removed,
dissected free from connective tissue and nonparenchymal pulmonary
tissue, and cultured as explants for 48 h in serum-free Waymouth MB
752/1 medium with penicillin and streptomycin in 95%
O2-5%
CO2 at 37°C. During this time,
endothelial and blood cells do not survive. The explant tissue was then
harvested, and the cells were dissociated using a solution of
collagenase, trypsin, and deoxyribonuclease. Cells were suspended in
minimal essential medium (MEM) with 100 U/ml penicillin and 100 µg/ml kanamycin containing 10% fetal bovine serum (FBS). The mixed cell suspension was subjected to differential adhesion on plastic tissue culture dishes (Costar, Cambridge, MA), and fibroblasts were separated by their property of adhering to the plastic. Nonadherent cells were
washed off the plate. Alveolar macrophages are not present in fetal
lungs and were not a source of contamination of fibroblast cultures.
Fibroblasts were cultured at 37°C in MEM containing 100 U/ml
penicillin, 100 mg/ml kanamycin, and 2.5 mg/ml amphotericin B (PKA) and
10% FBS. All cultures contained 94 ± 2% (mean ± SE) fibroblasts as determined by morphological appearance under phase contrast microscopy.
Exposure to hyperoxia.
Control samples were kept in room air-5%
CO2 environment at 37°C. The
O2 tension in the media was
70-80 mmHg. Test samples were kept in a Plexiglas chamber on a
rocking platform (Bellco Glass, Vineland, NJ), maintaining a constant
environment of 95% O2-5%
CO2 at 37°C for various time
points studied. This maintained an
O2 tension of 480-520 mmHg in
the media. The pH was not significantly affected (7.30-7.34 in
controls vs. 7.28-7.31 in hyperoxia). Cell viability at each time
point was 97 ± 2% (mean ± SE) in both conditions measured by
trypan blue exclusion as described by Freshney (16).
Cell proliferation in hyperoxia.
Nonconfluent fibroblasts obtained within 12 h after adhesion on 60-mm
tissue culture plates were washed with warm phosphate-buffered saline
(PBS), and the medium was changed to either MEM + PKA + 10% FBS
(regular medium) or MEM + PKA + 10% carbon-stripped FBS (CSFBS;
carbon-stripped medium). This was done to control for any effects of
carbon-stripped serum on cell proliferation.
[Methyl-3H]thymidine
(2 µCi/ml; specific activity 6.7 Ci/mmol) was added to each plate.
Replicate samples were kept in separate incubators at 37°C and were
exposed to normoxia (room air-5%
CO2) or hyperoxia (95%
O2-5%
CO2) for various periods of time
(2-96 h) before full confluence. Then the plates were washed with
PBS, and the cell monolayer was released from the plate with 1 ml of
0.05% trypsin treatment for 15 min. A fraction of this was taken for
counting in the hemocytometer, and the rest was used for the assay of
[3H]thymidine
incorporation as described by Smith et al. (28). Briefly, 5 ml of
absolute methanol were added to precipitate DNA. After refrigeration
overnight, the material was centrifuged at 2,000 g for 20 min, and the pellet was
washed with 5 ml of methanol and was recentrifuged. The supernatant was
decanted, and the pellet was solubilized in 1 ml of a solution of 2%
Triton X-100 and 2% sodium dodecyl sulfate (SDS). This was then mixed
with 10 ml of Opti-Fluor liquid scintillation cocktail and was counted
in a Beckman Scintillation Spectrometer. The results were expressed as
counts per minute (cpm) per 106
cells.
In three separate experiments, we studied the effect of 48 h of
exposure to O2 or room air on
confluent cultures of fibroblasts. Thymidine incorporation was studied
as described above.
RNA extraction and Northern analysis.
Confluent fibroblasts obtained 36-48 h after adhesion on 60-mm
tissue culture plates were washed with warm sterile PBS, and the medium
was changed to MEM + PKA + 10% CSFBS. Replicate samples were kept in
separate incubators and were exposed to room air-5% CO2 or hyperoxia (95%
O2-5%
CO2) for various time points. At
the end of each time point, control and test cultures were washed with
PBS, and total RNA was isolated by extraction in guanidium isothiocyanate using the method of Chomczynski and Sacchi (9). For RNA
electrophoresis, 5 µg of total RNA were loaded per lane as measured
by absorbance at 260 nm. Ethidium bromide staining was used to assess
the uniformity of loading per lane and the intact nature of the RNA.
The 18S and 28S ribosomal bands served as molecular size markers. The
RNAs were then separated for Northern blot analysis on 1% agarose gels
containing 5% formaldehyde and were transferred to a nylon membrane in
20× 3.0 M NaCl and 0.3 M sodium citrate (SSC). To measure
collagen message, we used a 1.8-kilobase (kb) EcoR I
fragment of a cDNA probe complementary to the mature protein coding
region of the
1(I)-procollagen
gene and a 0.7-kb EcoR I/Hind III fragment of
plasmid Hf934 for the
1(III)-procollagen probe
(generous gifts from Dr. B. E. Kream). A
-actin cDNA (generous gift
from Dr. J. Pachter) was obtained to serve as the background message
expression of a "housekeeping" gene. Probes were labeled with
32P by random priming and were
used for Northern blot analysis. After prehybridizing with 60%
formamide, 6× SSC, 5× Denhardt's reagent, and 0.2% SDS
with 100 µg/ml denatured salmon sperm DNA, hybridization was
performed at 42°C in a solution containing 50% formamide, 5×
SSC, Denhart's reagent, 0.2% SDS, 10% dextran sulfate, and 1 × 106 cpm/ml labeled cDNA probe. The
blots were washed at room temperature one time in 1× SSC and
0.1% SDS followed by three washes at 68°C in 0.2× SSC and
0.1% SDS. Autoradiography or
-scanning was carried out to obtain an
optimal signal for comparison.
Northern analysis of mRNA from nonconfluent and confluent
fibroblast cultures.
In some experiments, fibroblasts isolated within 12 h after adhesion on
plastic tissue culture plates were washed with PBS, and the cells were
incubated in MEM + PKA + 10% CSFBS. This time was designated 0 h for
these experiments. Replicate samples were kept in separate incubators
at 37°C and were exposed to room air-5% CO2 or hyperoxia (95%
O2-5%
CO2) for various times
(2-120 h). These cultures were initially nonconfluent and attained
full confluence between 24 and 48 h. No changes were made in the media
during the course of these experiments. At each specified time,
appropriate control and test cultures were harvested for RNA isolation
and Northern analysis as described above. These experiments were
different in that the time of initial plating of fibroblasts was taken
as 0 h to study the effects of O2
on nonconfluent cultures, whereas in other experiments involving RNA or
protein analyses, to study the effects of O2 on confluent
cultures, 0 h was the time at which cultures had reached complete
confluence.
Collagen protein synthesis.
Experiments were done in parallel to those mentioned above for mRNA
assessments with confluent fibroblast cultures. In the culture plates
that were used for collagen protein studies, 10 µCi/ml of
L-[5-3H]proline
(specific activity 25 Ci/mmol) were added to the culture media 2 h
before completion of the time period. At the end of the study period,
medium was removed from the cell layer and was placed in a glass tube.
The cells in each dish were scraped into 1 ml of homogenization buffer
[1 M NaCl, 50 mM
tris(hydroxymethyl)aminomethane · HCl (pH 7.4), 2.5 mM EDTA, 1 mM N-ethylmaleimide, and
0.2 mM phenylmethylsulfonyl fluoride] and were added back to
their respective medium. Each sample was adjusted to 15%
trichloroacetic acid (TCA) and was sonicated two times for 10 s on ice.
Precipitates were collected by centrifugation at 4,000 g for 20 min, washed one time with
10% TCA and one time with 10 mM potassium acetate in absolute ethanol,
and dissolved in 0.5 M NaOH. The incorporation of
[3H]proline into
collagenase-digestible and noncollagen protein was determined using
purified bacterial collagenase by the method of Peterkofsky and
Diegelmann (24). The amount of radioactivity solubilized by collagenase
is a measurement of the collagen synthesized. The remaining
radioactivity in the precipitate represents the noncollagen protein
synthesized. The percentage of collagen present in a portion of
analyzed protein substrate was determined on the basis of the following
calculations from Diegelmann and Peterkofsky (13): percentage of
collagen synthesis = [cpm in collagen digest/(cpm in residue × 5.4) + (cpm in collagen digest)] × 100. DNA content was determined using
diamidinophenylindole to correct for cell numbers in each dish (8).
Results are shown as the percentage of collagen synthesis per microgram
of DNA.
To further delineate the effects of hyperoxia on
1(I)-,
2(I)-, and
1(III)-net collagen production,
we used SDS-polyacrylamide gel electrophoresis (PAGE) of labeled
proteins extracted from fibroblasts. Experiments were done similar to
those mentioned above for RNA measurements except that tritiated
proline was added to the culture media for 24 h of labeling. After the
end of hyperoxic exposure at 24 and 48 h, the media were collected
separately. One milliliter of medium from each condition was taken and
concentrated using a Centricon 100 concentrator (Amicon, Beverly, MA)
spun at 1,000 revolutions/min (rpm) for 6 h. The 100 µl concentrate was acidified to a final concentration of 0.5 M acetic acid solution. This was digested with pepsin (2 mg/ml final concentration) for 6 h at
16°C. The acetic acid was neutralized with equimolar solution of
NaOH, and a repeat Centricon spin at 1,000 rpm was done for 1 h to get
a final volume of ~75 µl. This was mixed with protein sample buffer
containing 5 M urea and was boiled for 5 min. This was loaded on a 5%
polyacrylamide gel, and electrophoresis was performed using the Sykes
et al. modification (30) of the Laemmli method (23). Briefly, this
involved changing to reducing conditions with
-mercaptoethanol after
an initial run to delineate the separation of the subunits of type I
and III collagens. The gel was then fixed with 10% (vol/vol) Glacial
acetic acid and 30% (vol/vol) methanol and was kept in EnHance for 1 h. After immersing in cold water for 0.5 h and drying the gel, the
radioactive emission by tritiated proline incorporated in the collagen
was captured on a Kodak X-OMAT AR photographic film after an 8-day
exposure at
80°C (20, 21).
Statistics.
All data are means ± SE of three to five experiments. Statistical
significance was determined using the Student's
t-test or analysis of variance with
post hoc Bonferroni/Dunn test as appropriate. Correlations were
determined using Spearman correlation. Differences were considered
significant at P < 0.05.
Materials.
Timed pregnant Sprague-Dawley rats were purchased from the Charles
River Breeding Laboratories (Wilmington, MA). All culture media were
purchased from GIBCO-BRL (Grand Island, NY). Culture supplies and
plastic ware were obtained from Costar (Cambridge, MA). Collagenase and
trypsin for cell dissociation were obtained from Worthington (Freehold,
NJ). Radioisotopes were purchased from New England Nuclear (Boston,
MA). Opti-Fluor was bought from Packard (Meriden, CT). FBS was
purchased from Hyclone (Logan, UT). All other chemicals and reagents,
including purified bacterial collagenase for the assay, were purchased
from Sigma Chemical (St. Louis, MO). FBS (Hyclone lot no. 11112207) was
carbon stripped by the method of Yoshizato et al. (34) as modified by
Tanswell et al. (31).
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RESULTS |
Cell viability.
When cells and tissues are exposed to high concentrations of
O2, they exhibit toxic effects
leading to cell death (3, 17). However, fibroblasts from fetal rat
lungs in these experiments were resistant to the cytotoxic effects of
hyperoxia, as their cell viability was 97 ± 2% at all time points
studied up to 5 days. The mRNA levels for the housekeeping gene
-actin also remained fairly constant (see below), indicating that
these cells were maintaining their vital function.
Cell proliferation.
Data for the effect of hyperoxia on
[3H]thymidine
incorporation by fetal lung fibroblasts cultured in media containing
either regular or CSFBS are shown in Fig.
1. In nonconfluent fibroblasts cultured in
regular medium (MEM + PKA + 10% FBS) and exposed to room air
conditions,
[3H]thymidine
incorporation per 106 cells
increased 67-fold, from 1,901 ± 252 cpm at 2 h to 128,729 ± 1,462 cpm at 96 h. At 96 h of exposure to hyperoxia, the
[3H]thymidine
incorporation was attenuated over fivefold to 23,718 ± 1,039 cpm in
nonconfluent fibroblasts cultured in regular medium (P < 0.0001).

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Fig. 1.
Hyperoxia inhibits proliferation of fetal rat lung fibroblasts. Cells
were cultured in either media containing charcoal-stripped fetal bovine
serum or regular fetal bovine serum.
[3H]thymidine
incorporation was expressed as counts per minute (cpm) per
106 cells. Data are means + SE of
7 samples. Proliferation of cells exposed to 95%
O2 (O2) was significantly
inhibited compared with room air-exposed cultures (RA) at 24, 72, and
96 h of exposure (* P < 0.05, # P < 0.01, and
§ P < 0.001, respectively). Cell proliferation in carbon-stripped medium was lower
than regular medium but the percentage of inhibition of proliferation
in hyperoxia was similar in both media at all time points studied.
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Nonconfluent cultures of fibroblasts incubated in carbon-stripped media
(MEM + PKA + 10% CSFBS) in room air exhibited a 10-fold increase in
proliferation from 2 to 96 h. In nonconfluent fibroblasts cultured in
carbon-stripped media, there was a similar significant decrease in
[3H]thymidine
incorporation in hyperoxia-exposed cells compared with normoxia-exposed
cells (P < 0.0001). Cell
proliferation as measured by
[3H]thymidine
incorporation was significantly lower in fibroblasts incubated in room
air with carbon-stripped media compared with regular media at 24, 72, and 96 h (P < 0.001).
Cell proliferation was significantly decreased in the hyperoxia-exposed
fibroblast cultures incubated in both regular and carbon-stripped media
at 24 (P < 0.05), 72 (P < 0.01) and 96 (P < 0.001) h. There was no effect
of 48 h of exposure to hyperoxia on cell proliferation in confluent
fibroblast cell cultures (regular medium: room air 6,399 ± 600 cpm/106 cells vs.
O2 5,912 ± 37 cpm/106 cells,
P = not significant; carbon-stripped
medium: room air 5,620 ± 935 cpm/106 cells vs.
O2 3,623 ± 78 cpm/106 cells,
P = not significant).
Time-dependent decrease in procollagen mRNA levels in hyperoxia.
In confluent fibroblast cultures, we found a decrease in the
steady-state mRNA levels of both
1(I)- and
1(III)- procollagens with
hyperoxia exposure at all time points studied, starting as early as 4 h
(data not shown) and continuing up to 48 and 120 h (Table
1). Intact and comparable RNA loading per
lane was confirmed by ethidium bromide staining and also by
-actin
mRNA levels that did not change as a result of hyperoxia. Densitometric
analysis of the autoradiographs was done, and the steady-state mRNA
levels of
1(I)- and
1(III)-procollagens in room air
controls were normalized to 100% for comparison. The mRNA levels of
1(I)-procollagen in fibroblasts
exposed to hyperoxia were 65 and 36% of controls kept in room air at
48 and 120 h, respectively, as shown in Table 1 (P < 0.01). Similarly, the
steady-state mRNA levels of
1(III)-procollagen in
fibroblasts cultured in hyperoxia were 56 and 23% of controls kept in
room air at 48 and 120 h, respectively
(P < 0.01). There was a significant
inverse correlation between steady-state mRNA levels and time of
exposure to hyperoxia for
1(I)-procollagen (r =
0.904,
P < 0.001) and
1(III)-procollagen
(r =
0.971,
P
0.001). This was a selective
effect on mRNA collagen expression because the steady-state levels of
-actin mRNA did not change.
Time-dependent changes in procollagen mRNA levels in room air.
Interestingly, there was an increase in the steady-state levels of
1(I)-procollagen mRNA in
fibroblasts cultured in room air in the first 48 h (Fig.
2A,
lanes
1, 3,
and 5). A similar increase was seen
in levels of
1(III)-procollagen
mRNA (data not shown). This was followed by a decrease in mRNA levels
for these procollagens at 120 h in culture (Fig.
2B,
lane
3, and Fig. 3, lane
3). The decrease in expression of
procollagen mRNA at 120 h may be a reflection of the feedback
inhibition of gene transcription by collagen peptides that has been
previously shown by others (10, 33).

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Fig. 2.
Time-dependent effects of hyperoxia on levels of mRNA for
1(I)-procollagen. Fibroblast
cultures after initial plating became confluent after 24-48 h in
culture. Five micrograms of total RNA were loaded in each lane.
Autoradiogram of Northern blot using rat
1(I)-procollagen
[32P]cDNA probe
(top). The same membrane was
stripped and probed with
[32P]cDNA probe for
-actin (bottom).
A: representative autoradiograph of
steady-state levels of mRNA for
1(I)-procollagen in
nonconfluent fibroblast cultures (early time points 4-48 h). There
is a time-dependent increase in steady-state mRNA levels in room air
(R). There is an increase in mRNA levels in hyperoxia (O) over 24 h,
followed by a decrease by 48 h. At all times, mRNA levels are lower in
cultures exposed to O2 compared
with the corresponding cultures incubated in room air. No significant
changes in -actin levels are seen.
B: representative autoradiograph of
steady-state levels of mRNA for
1(I)-procollagen in confluent
fibroblast cultures. There is a decrease in mRNA levels in room air
from 96 to 120 h. mRNA levels in fibroblasts exposed to hyperoxia
continue to decrease with time. There are no significant changes in
-actin mRNA levels.
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Fig. 3.
Representative autoradiograph of steady-state levels of mRNA for
1(III)-procollagen expressed by
fetal rat lung fibroblasts exposed to hyperoxia. Equal amounts (5 µg)
of total RNA were loaded in each lane. Northern blot was performed
using a rat 1(III)-procollagen
[32P]cDNA probe and
autoradiography (top). The same
membrane was stripped and was probed with a
[32P]cDNA probe for
-actin (bottom). The actin
message remained relatively constant, but the
1(III)-procollagen message was
decreased in hyperoxia (O2) at
48 h compared with room air (RA). A greater decrease was noted in
1(III)-procollagen mRNA at 120 h. mRNA levels in RA show a decrease at 120 h compared with 48 h.
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Changes in procollagen mRNAs in nonconfluent and confluent
fibroblast cultures.
When nonconfluent fibroblasts soon after adhesion were exposed to room
air or hyperoxia, the procollagen mRNA levels in hyperoxia-exposed cultures were lower than the room air-exposed cultures at each time
point (Fig. 2A). In the first 24 h
of culture, before the fibroblasts became confluent, the procollagen
mRNA levels in hyperoxia increased to a lesser degree over time than
the corresponding cultures exposed to room air. However, on continued
culture of fibroblasts from 48 to 120 h (Fig.
2B), after full confluence was
attained, the procollagen mRNA levels in hyperoxia were markedly reduced compared with the corresponding room air cultures (Fig. 2,
A,
lanes
5 and
6, and
B).
Percentage of collagen protein synthesis.
To demonstrate that the decreases in mRNA levels were reflected by a
concomitant decrease in net collagen protein production in the
fibroblasts, we studied the incorporation of
[3H]proline in
collagen protein. The percentage of collagen synthesis at each time was
calculated according to Peterkofsky and Diegelmann (24). Figure
4 shows the percentage of collagen
synthesis at 24 h of hyperoxia exposure. We observed a 30% decrease in
the percentage of net collagen protein synthesis in hyperoxia compared with room air (P < 0.05).

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Fig. 4.
Hyperoxia inhibits net collagen production by fetal rat lung
fibroblasts after 24 h of exposure. Tritiated proline incorporation in
collagenase-digestible proteins was measured, and percentage of
collagen synthesis was calculated as described in
METHODS AND MATERIALS. Results are
shown as percentage of collagen synthesis per microgram (mcg) of DNA.
There was a significant decrease in percentage of collagen synthesis
(* P < 0.05).
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To determine if this was a specific inhibition in collagen synthesis
versus a nonspecific inhibition of total protein synthesis, we compared
the collagenase-digestible protein fraction with the noncollagenase-digestible fraction, which is representative of general
protein synthesis. Figure 5 shows the
percent change in each fraction with increasing duration of exposure to
hyperoxia, considering 0 h levels to be 100%. The percent decrease in
collagen protein was highly correlated to time of hyperoxia exposure
(r =
0.944,
P < 0.001). The absolute decrease at
12 and 24 h of exposure was also statistically significant
(P < 0.05). Conversely, noncollagen
protein levels showed a positive correlation with time of hyperoxia
exposure (r = +0.916,
P < 0.001). The absolute increase in
noncollagen proteins at 24 h of hyperoxia was also statistically
significant (P < 0.05). This
confirmed our observation that the time-dependent effect of hyperoxia
was specific for collagen and not a nonspecific or toxic decrease in
total cellular protein synthesis.

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Fig. 5.
Percentage change in net production of collagen and noncollagen
proteins with increasing time of exposure to hyperoxia. Tritiated
proline incorporation in collagenase-digestible proteins representing
total collagen proteins was compared with its incorporation in
collagenase-nondigestible proteins representing the noncollagenous
proteins. The levels of both fractions were normalized to 100% at 0 h
of exposure to hyperoxia. Data are means + SE of 3-5 experiments.
There was a significant inhibition of collagen synthesis at 12 and 24 h
of exposure (* P < 0.05). In
contrast there was a significant increase in noncollagen protein
synthesis at 24 h (# P < 0.05).
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As shown in Fig. 6, we also measured net
collagen production using SDS-PAGE of labeled cellular proteins to
confirm these effects and to further examine the effects on net
production of
1(I)- and
2(I)-collagens by fetal lung
fibroblasts exposed to hyperoxia for 24-48 h. We found a
significant decrease in net production of both collagens by fibroblasts
exposed to hyperoxia at both 24 and 48 h. We were unable to detect
1(III)-collagen proteins in
either control or experimental cultures using this method.

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Fig. 6.
Fluorogram of gel after SDS-polyacrylamide gel electrophoresis of
tritiated proline-labeled collagen secreted from fetal rat lung
fibroblasts incubated in room air (RA) or hyperoxia
(O2) for 24 and 48 h. Top
arrowhead, 1(I)-collagen;
bottom arrowhead,
2(I)-collagen. Type III
collagen was not detected even after concentration of the medium.
Significant inhibition of type I collagen subtypes
1(I) and
2(I) secretion into the medium
is demonstrated. Figure shown is representative of 1 of 3 experiments.
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DISCUSSION |
The pulmonary response to hyperoxic exposure is characterized by
extensive injury to airway, parenchymal, and vascular structures leading to inflammation and tissue remodeling that may ultimately result in complete repair or fibrosis (12). This situation is further
influenced by immaturity of specific lung cells in the premature infant
that develops bronchopulmonary dysplasia (6). Few studies have
addressed the direct effects of O2
on specific lung cells. Those studies dealing with fibroblasts have
utilized established cell lines (4) or primary cultures of cells
obtained from adult animals (29). No study before this has reported on the direct effects of hyperoxia on cellular proliferation or expression of collagen synthesis in primary cultures of fibroblasts derived from
fetal rat lungs. This model may be more relevant to an understanding of
the biomolecular events involved in hyperoxic injury to premature lungs.
Hyperoxia inhibits fibroblast proliferation.
We have shown that hyperoxia decreases proliferation of cultured fetal
rat lung fibroblasts after 24-96 h exposure. Cell proliferation in
hyperoxia was ~10-30% of that in room air, given optimal
temperature, pH, and CO2
environment. This inhibitory effect was seen with both carbon-stripped
and regular media. Experiments done in parallel with regular and
carbon-stripped media show that carbon stripping (which removes
hormones such as glucocorticoids that influence cellular proliferation
and collagen expression) decreases proliferation, but the effects of
hyperoxia are still significant in both media conditions studied. We
conclude that hyperoxia does inhibit fibroblast proliferation.
The decrease in fibroblast proliferation with hyperoxia concurs with
other reports of similar inhibition noted in fibroblast cell lines and
primary cultures from mature adult lungs (1, 17). However, in vivo
exposure of lungs to elevated levels of O2 leads to increased fibroblast
proliferation (12). Shapiro et al. (26) reported that exposure of lung
slices in organ culture to hyperoxia for 3 days also caused increased
proliferation of parenchymal cells that returned to baseline after 7 days of exposure. The specific cells affected, however, were not
described in that study. Kelleher et al. (19) recently showed that lung
fibroblasts isolated from rats that were exposed to hyperoxia
proliferated more rapidly than fibroblasts obtained from rats exposed
to room air. The fibroblasts isolated from hyperoxia-exposed animals
were more sensitive to the proliferative effects of insulin-like growth factor I, and these cells had a higher expression of c-Ha-ras, which is
an essential protein for progression through the cell cycle (19).
Unfortunately, these experiments were performed on cells isolated after
the tissue was exposed, which makes it difficult to exclude primary or
secondary influences of other cell types that were present. Moreover,
these cells were cultured in serum that may contain hormones and growth
factors that might confound the interpretation of these results.
Nevertheless, these studies indicate that cell-cell interactions with
neighboring cells are important in the in vivo responses.
Hyperoxia decreases levels of procollagen mRNA in confluent cultures
of fibroblasts.
Our experiments demonstrate that hyperoxia results in a time-dependent
decrease in steady-state mRNA levels for both types
1(I)- and
1(III)-procollagens in
confluent fetal lung fibroblast cultures. This is a novel finding
because previous studies by Housset et al. (17) with mixed cell
cultures have shown that exposure to elevated
O2 levels leads to an increase in
1(I)-procollagen mRNA.
Similarly, Armstrong et al. (2) demonstrated that oxidant exposure of
rats increased the lung content of type I collagen mRNA. This
discrepancy between the responses of primary cell cultures and mixed
lung cell cultures can be reconciled if it may be suggested that other
cells, especially alveolar macrophages, and proinflammatory cytokines
[e.g., tumor necrosis factor (TNF)-
, transforming growth factor (TGF)-
, and interleukins] are involved in mediating the effects of hyperoxia in vivo. Recent evidence has shown that alveolar macrophage-depleted rats have a significantly higher tolerance to
hyperoxia than controls (5). Also, TNF-
has been shown to enhance
susceptibility of lung cells to O2
toxicity, and administration of anti-TNF antibodies protects against
this toxicity (18).
Biphasic time-dependent effects in steady-state collagen mRNA in
fibroblasts cultured in room air.
We found an increase in the levels of
1(I)-procollagen mRNA in lung
fibroblasts cultured in room air over the first 48 h after plating,
which was followed by a decrease in steady-state levels at ~120 h
after plating (Fig. 2B). The same
was true for
1(III)-procollagen
mRNA (Fig. 3). Whereas it is possible that this later decrease in
levels of mRNA is due to the time-dependent effects on cell
proliferation, this later decrease in mRNA occurs well after the
fibroblasts have reached confluence, making this possibility less
likely. A more likely explanation for these findings is that there is
an initial time-related increase in the mRNA levels of procollagens
when fibroblasts are freshly cultured; once cells have reached
confluence and enough collagen protein has been translated and
secreted, then feedback inhibition of collagen expression would lead to
reduced steady-state levels of procollagen mRNA. The role of collagen
peptides on pretranslational regulation of collagen synthesis has been
well documented (10, 33). Comparison of results from different studies
must take these effects of time and confluence of cultures into
account.
Effect of hyperoxia on collagen expression is pretranslational.
In our study, the decrease in mRNA levels was detectable by 4-6 h
exposure of fibroblasts to high O2
levels and became progressively more pronounced with increasing times
of exposure up to 5 days without affecting the cell viability. The
steady-state levels of
-actin mRNAs were not affected, suggesting
that the effect on procollagen mRNAs was selective and was not a global
shut down of cell machinery due to the toxic effects of
O2. The demonstration that there
was a specific decrease in net collagen production, with respect to
noncollagenous proteins (Fig. 6), supports our conclusion that the
changes shown are specific and not general. In fact, the increase in
noncollagen protein synthesis at 24 h of exposure indicates that the
cellular mechanisms that support protein synthesis remain intact. We
therefore conclude that hyperoxia directly effects a decrease in
collagen gene expression at the pretranslational level in fetal rat
lung fibroblasts.
The mechanism by which hyperoxia directly affects fibroblast collagen
expression is unknown. It has recently been suggested by Duncan et al.
(14) that the transcription factor nuclear factor-1, which is
influenced by the cell redox state, may regulate the promoter for both
collagen types I and III. Falanga et al. (15) have shown that low
O2 tensions increase mRNA levels
of
1(I)-procollagen in
fibroblast cell cultures. Our finding that high
O2 tensions decrease the levels of
both
1(I)- and
1(III)-procollagen mRNAs in
fibroblast cultures may be another clue that changes in the cell redox
state may regulate collagen expression.
In conclusion, we have shown that the direct effects of hyperoxia on
fetal rat lung fibroblast cultures are
1) a decrease in proliferation and
2) a pretranslational decrease in
types I and III procollagen expression. Because the opposite effects, i.e., increased proliferation and collagen expression, are seen in vivo
and in tissue or organ cultures, we speculate that other cell types
(alveolar macrophages) or cytokines (e.g., TNF-
, TGF-
, and
interleukins) may mediate the profibrotic effects of hyperoxia.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Roger Thrall and Alex Lichtler for helpful review of
the manuscript. We are also grateful to Drs. Barbara Kream and Joel
Pachter for the cDNA probes and Eileen Roark and Penny Kelly for
technical support and assistance.
 |
FOOTNOTES |
This work as supported in part by grants from the Charles H. Hood
Foundation (to M. J. Kresch) and Patterson trust (to M. J. Kresch).
Address for reprint requests: N. Hussain, Dept. of Pediatrics, Univ. of
Connecticut School of Medicine, Mail Code #2203, Farmington, CT 06030.
Received 13 March 1996; accepted in final form 17 June 1997.
 |
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