Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027
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ABSTRACT |
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The final stage of lung
development, alveolarization, continues after birth in humans and
rodents. Clinical interventions, such as oxygen therapy, in the first
week of life can adversely impact alveolar formation. We
compared alveolarization in the rat neonate under normal vs. hyperoxic
conditions, examining gelatinase, transforming growth factor (TGF)-,
and the protease urokinase-type plasminogen activator (uPA) activities
in whole lung and cultured type II alveolar epithelial cells
(AEC2). The dynamic induction of gelatinase, TGF-
, and uPA
activities seen in neonatal lungs during the first days of life was
significantly impacted by hyperoxia. In whole lung, gelatinase and
TGF-
activities were increased, while uPA activity was decreased. At
the level of the epithelium, AEC2 isolated from hyperoxic rat pups
early in life secreted less active TGF-
, less active gelatinases,
and less active uPA than control neonatal AEC2. AEC2 from
hyperoxic pups also expressed increased levels of proliferating cell
nuclear antigen early in life compared with control neonatal AEC2,
suggesting that oxygen-induced proliferation and/or repair were
occurring. The developmental profile of neonatal lung was perturbed
within a day of initiating oxygen treatment, suggesting that putative
palliative treatments should be coadministered with oxygen therapy.
matrix metalloproteinases; active transforming growth factor-; gelatinase A; gelatinase B; urokinase-type plasminogen activator; type
II alveolar epithelial cells
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INTRODUCTION |
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ALVEOLARIZATION IS THE
FINAL STEP in lung morphogenesis. It is initiated late in
gestation and, in humans as well as rodents, continues after birth.
Premature human infants treated with oxygen and mechanical ventilation
during this critical window of lung development are at risk for
bronchopulmonary dysplasia (2), a chronic condition
characterized by alveolar hypoplasia and fibrosis (28).
Thus neonatal clinical interventions such as oxygen therapy, although
necessary to sustain life, may also adversely impact alveolarization.
Diminished alveolar development is a uniquely neonatal response to
hyperoxia (33). We have previously shown that the lungs of
neonatal rats exposed to hyperoxia during the first week of life show
hypercellularity and edema associated with sequential induction of
TNF-, followed by IL-6 (5). Although the infiltration
resolves after removal of oxygen, long-term effects may include
decreased alveolar surface area, enlargement of alveolar ducts, and
increased parenchyma (25).
Transforming growth factor (TGF)- is necessary for normal lung
development, as suggested by the neonatal-lethal phenotype of TGF-
3
knockout mice, which die in the first 24 h of life of pulmonary
failure. Their lungs show some phenotypic similarities to hyperoxic
lungs with alveolar hypoplasia and mesenchymal thickening (22). Metalloproteinases and urokinase-type plasminogen
activator (uPA) have also been implicated in neonatal lung development, since they are expressed in tissues undergoing active remodeling (10, 17, 29, 30). There is considerable cross talk among TGF-
, metalloproteinases, and uPA. TGF-
can be activated by metalloproteinases (35) and may, directly or indirectly,
activate gelatinases (27). TGF-
is also activated by
the protease plasmin, which is activated by uPA (23). uPA
can also activate metalloproteinases (3, 23). On the basis
of these observations, we hypothesized that TGF-
and potential
activators, such as uPA and metalloproteinases, would be
developmentally regulated in the lung during alveolarization and would
be likely targets of hyperoxic injury.
The type II alveolar epithelial cell (AEC2), which plays a pivotal role
in alveolar basement membrane remodeling after injury and conceivably
during alveolarization, constitutively secretes active TGF-3
(6), uPA (19), and gelatinases A and B
(7, 30). The effects of hyperoxia on TGF-
activation
and gelatinase activity have been characterized during the remodeling
phase after hyperoxic injury repair in adult rat AEC2 (6,
7), but analogous profiles have not been determined for neonatal
AEC2 from rats exposed to hyperoxia during alveolarization. Similarly,
the activities of TGF-
, gelatinase, and candidate activators, such
as uPA, have not been measured in the neonatal lung during normal
alveolarization vs. hyperoxic intervention.
Using our neonatal rodent hyperoxia model (5), we measured
and compared the dynamic expression and activity of TGF-, the gelatinases, and uPA during the course of early alveolarization under
normoxic vs. hyperoxic conditions. The rationale for using cultured
neonatal AEC2 was to compare the neonatal AEC2 hyperoxic response with
that of the adult hyperoxic AEC2. The alveolar environment differs
substantially between the adult and neonate. In the adult model of
hyperoxia, the damage is inflicted on a relatively quiescent alveolar
epithelium, whereas in the neonatal model the damage occurs against a
background of active epithelial remodeling and alveolarization. Whole
lung was also examined at specific key times after birth to establish
the milieu of the AEC2.
We demonstrate developmental increases in TGF-, gelatinase, and uPA
activities in whole lung during the first week of life and show that
hyperoxia significantly affects these parameters. Hyperoxia-mediated
changes in TGF-
, gelatinase, and uPA activities were also detected
in neonatal AEC2. These specific changes to whole lung and AEC occur
very early on during the hyperoxic regimen, suggesting a very narrow
window for intervention may exist.
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METHODS |
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Oxygen treatment. Litters of rat pups, born within 2 h of each other, were pooled and divided into two groups. One group was placed in a 90 × 42 × 38-cm Plexiglas chamber, exposed to >90% humidified oxygen for 9 days, and kept in room air thereafter. The second group was maintained in room air. The mothers of the control and hyperoxic litters were exchanged daily to minimize maternal oxygen toxicity. Five individual hyperoxic exposures were performed. Animal-to-animal variation in response to hyperoxia was apparent by day 6, with ~ 30% mortality on or after day 6.
Isolation and culture of neonatal AEC2. Neonatal AEC2 were isolated from lung on days 3, 6, and 10 after birth, as preliminary data from whole lung indicated that significant events occurred at or about days 3 and 6 and resolved by day 10. The method used was elastase digestion followed by differential adherence on IgG plates, as described by Dobbs et al. (13) with additional modifications for neonates as described by Griese et al. (18). We routinely added an additional step in which the cells panned from the IgG plates were further purified by an extra 1 h of adherence on plastic in the presence of 10% FCS to remove any remaining fibroblasts. A pool of 6-8 pups was used for each condition, yielding ~2 × 106 cells/animal in the final fresh isolate, which was plated in Primaria six-well plates and on a chamber slide for purity assessment. The culture medium was HEPES-buffered DMEM (Sigma, St. Louis, MO) with 10% FCS and antibiotics. After overnight attachment, the cultures were washed to remove unattached cells, and DMEM plus 0.1% BSA was added for autocrine secretion studies. We estimated purity after 24 h of attachment by immunostaining acid-alcohol fixed cells with an antibody to surfactant protein C (Santa Cruz Biotechnology, Santa Cruz, CA); it was 80-85%. Isolations from 3-, 7-, and 10-day-old pups were of similar purity.
Collection of conditioned medium from AEC2.
The serum-containing medium was withdrawn after the AEC had fully
attached, and the cultures were washed to remove unattached cells and
debris. DMEM containing 0.1% BSA was added, and the cultures were
returned to the incubator for 24 h. The medium was removed,
centrifuged to remove cells, and frozen at 70°C for TGF-
, uPA,
and gelatinase activity measurement. The cells corresponding to the
conditioned medium were trypsinized and counted.
Preparation of conditioned medium from whole lung.
Lung tissue was dissected from the airways, blotted, weighed, and
placed in iced PBS. It was then chopped into small pieces, extensively
washed to remove blood, and homogenized by Polytron on ice in DMEM-F12
medium containing 2% BSA, 2.5 µl/ml 0.2 M PMSF, 5 µl/ml aprotinin,
and ~10 trypsin inhibitor units/ml (4). In samples that
were to be used for uPA estimation, antiproteases were omitted. The
homogenates were incubated at 37°C for 1 h and centrifuged at
14,000 g for 10 min at 4°C (4). The
supernatants were frozen at 70°C until assay.
Preparation of lung homogenates for matrix metalloproteinase
activity.
Lung tissue was dissected and washed as above then homogenized by
Polytron on ice in 50 mM Tris · HCl, pH 7.4, containing 1 mM
monothioglycerol, centrifuged at 2,000 g for 10 min at
4°C, and frozen at 20°C (14).
Preparation of whole lung lysates for Western blotting.
Lung tissue was dissected and washed as above. Pools consisting of
three lungs from each time point were homogenized in lysis buffer at
4°C with a Polytron homogenizer (8). The
homogenates were centrifuged at 14,000 g for 10 min at
4°C, and the supernatants removed, assayed for protein levels, and
frozen at 20°C.
Lung lavage.
The lungs were lavaged to total lung capacity six times with ice-cold
PBS through the trachea while still in the animal. The washes were
pooled, centrifuged at 430 g at 4°C for 5 min to pellet any cells, and frozen at 70°C for later analysis.
Measurement of matrix metalloproteinase-9 and matrix metalloproteinase-2 activity. We measured gelatinase activity in conditioned medium from cultured "hyperoxic" and control neonatal AEC, as well as in whole lung, using the Biotrak assay systems from Amersham-Pharmacia Biotech UK (Little Chalfont, UK) according to the manufacturer's instructions. The assay uses the proform of a detection enzyme that can be activated by captured active matrix metalloproteinases (MMP) into an active detection enzyme through a single proteolytic event. The assays are done in 96-well plates, and color product is detected using a plate reader. MMP is calculated relative to standards provided with the kit and corrected for cell number or lung weight. The advantage of this assay method over gelatin zymography is twofold: sensitivity down to ~0.125 ng MMP/ml and the ability to quantitate active MMP levels.
Western blotting of proteins. Western analysis was performed on cell lysates as described by Bui et al. (8), using 20 µg of protein/lane. Control and hyperoxic samples were loaded onto the same 15-well commercially prepared gel (Novex/Invitrogen, Carlsbad, CA). We confirmed equal loading by reprobing the blots with an antibody to actin. We detected proteins of interest with the use of horseradish peroxidase-linked secondary antibodies and the enhanced chemiluminescence system following the manufacturer's instructions (Amersham, Arlington Heights, IL). Blots were repeated with lysates from different pools of control and hyperoxic animals to confirm differences. Antibodies to MMP-9 were from Chemicon (Temecula, CA); antibodies to proliferating cell nuclear antigen (PCNA) were from Santa Cruz Biotechnology. Antibodies to MMP-2, membrane-type (MT)-1-MMP, MMP-1, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were from Oncogene Research Products (Boston, MA). Antibody to actin was from ICN (Irvine, CA). Secondary antibodies were from Sigma. Blots were scanned with Adobe PhotoDeluxe Business Edition software and analyzed with Scion Image software.
TGF- activity.
We measured TGF-
activity by the plasminogen activator inhibitor
(PAI-1) luciferase assay, using mink lung epithelial cells stably
transfected with an expression construct containing a truncated PAI-1
promoter fused to a firefly luciferase reporter gene (1). The cells were generously provided by Dr. Dan Rifkin of New York University. Addition of active TGF-
to these cells results in a
dose-dependent expression of luciferase with a sensitivity of <3.6
pg/ml. Mink lung epithelial cells containing the PAI-1-luciferase construct were plated in 96-well plates, 1.6 × 104
cells/well, in DMEM with 10% FCS and allowed to attach for 3 h or
more. The serum-containing medium was then removed and replaced with
conditioned medium from neonatal AEC and neonatal lungs and assayed in
parallel with TGF-
standards. After 24 h of incubation, the
cells were washed, lysed, and assayed for luciferase activity with the
enhanced luciferase assay kit from Pharmingen (San Diego, CA) and a
Berthold luminometer (WALLAC, Gaithersburg, MD). Active TGF-
was
corrected for cell number in the case of neonatal AEC2, or lung weight
when lung homogenates were used.
TGF- isoform determination.
Conditioned medium from AEC2 was incubated for 2 h at room
temperature with saturating doses of neutralizing antibodies to TGF-
1 and TGF-
3 (R & D Systems, Minneapolis, MN), and the
active TGF-
levels were compared with untreated conditioned medium.
uPA activity. uPA activity was measured in lung-conditioned medium and conditioned medium from neonatal AEC2 using reagents from Chemicon. A chromogenic substrate is cleaved by active uPA to produce a color product, which is detected on a plate reader, achieving sensitivities of 0.626 units of uPA/ml. Active uPA was calculated relative to standards provided with the kit and corrected for cell number when AEC2 were used, or lung weight when lung homogenates were used.
Statistics. Results are expressed as means ± SD. Statistically significant differences in the mean values were analyzed using the Student's t-test, with P values <0.05 considered significant.
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RESULTS |
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Dynamic expression of MMP in neonatal lung in the first week of
life is disrupted by hyperoxia.
Western analysis of neonatal lung lysates shows that the expression of
the two gelatinases MMP-9 and MMP-2 and of MT-1-MMP, which can activate
MMP-2, increases in the first week of life (Fig.
1A). MMP-9 expression is
increased about fourfold in hyperoxic lysates on day 2 (P < 0.01) compared with control lysates, and increased expression is still detected at day 4 (P < 0.01) and day 6 (P < 0.01). MMP-2 expression is increased on days 2 and 4 (each P < 0.0125). MT-1-MMP expression is
also induced by hyperoxia, but later than the gelatinases, on day
8 (P < 0.05) and day 10 (P < 0.025). In contrast, interstitial collagenase
does not increase after birth and remains unaffected by hyperoxia,
suggesting that either hyperoxia affects the activity rather than the
expression of MMP-1 or that this metalloproteinase may not play a
significant role in the lung's early response to hyperoxia.
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Hyperoxia induces expression of TIMP-1 in neonatal lungs. TIMP-1, which preferentially inactivates MMP-9, is barely detectable in control neonatal lung lysates but is induced during the first week of life by hyperoxia at levels approximately threefold higher than control levels (P < 0.0025, n = 3). In contrast, TIMP-2, which preferentially inactivates MMP-2, is expressed from birth and is not induced in the first days after hyperoxia.
Hyperoxia increases gelatinase activity in early neonatal lungs.
Because of the induction of gelatinase expression seen in neonatal lung
after hyperoxia, we compared the respective gelatinase activities using
extracts from control vs. hyperoxic lungs (Fig. 2A, MMP-9; Fig. 2B,
MMP-2). Active MMP-9 or MMP-2 was measured on 96-well plates as a color
product proteolytically released by captured active enzyme. MMP-9
activity in hyperoxic lungs showed a twofold increase over control
values on day 2, followed by a threefold increase on
day 4, and returned to control values by day 10.
MMP-2 activity in hyperoxic showed a fourfold increase over
control values at day 2, decreasing to levels below control values on days 4, 6, and 8.
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Neonatal AEC2 from hyperoxic rat pups secrete significantly less
active gelatinases early in life than control AEC2.
In contrast to events seen in whole lung, cultured neonatal AEC2
isolated from hyperoxic animals showed a threefold decrease in
gelatinase activity when isolated early in life (Fig.
3). Both MMP-9 activity (Fig.
3A) and MMP-2 activity (Fig. 3B) in conditioned medium from hyperoxic AEC2 were lower than control values on day 3 of life. The effects of hyperoxia on active MMP-2
secretion by neonatal AEC2 were still apparent at day 6,
while the MMP-9 activity had returned to control values.
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Dynamic activity of TGF- in developing neonatal lung is
disrupted by hyperoxia.
The amount of active TGF-
recovered from normal rat lung increases
threefold during the first week of life (Fig.
4) as measured by PAI-1 induction of
luciferase activity in Mv1Lu cells (1). By day
6, a 1.5-fold induction of TGF-
activity is detected in hyperoxic lungs, and activity remains elevated through day
10. As these data are necessarily expressed as per milligram of
lung (due to the presence of 2% BSA in the homogenates), the decrease seen in TGF-
activity seen at day 2 may be artifactual,
due to the contribution of pulmonary edema to the lung wet
weight. The edema resolves after day 3 (5).
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Increased levels of active TGF- are recovered from the lung
washes of hyperoxic neonates.
Analysis of lung washes from control and hyperoxic rat pups lavaged to
total lung capacity six times with PBS shows a developmental activation
of TGF-
(Fig. 5), which reflects whole
lung TGF-
(Fig. 4). There is sixfold more active TGF-
recovered
from the lung washes of hyperoxic rat pups compared with control pups
during the first week of life, which persists for at least 4 days after removal from oxygen.
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Neonatal AEC2 from hyperoxic rat pups secrete significantly less
active TGF- early in life than control neonatal AEC2.
Aliquots of conditioned medium from neonatal AEC2 cultured from
hyperoxic vs. control rat pups were assayed for active TGF-
. Lungs
were removed from neonates on days 3, 6, and
10, and AEC2 were isolated by elastase digestion and IgG
selection (13, 18). Neonatal AEC2 from hyperoxic rat pups
secrete ~50% less active TGF-
than control AEC2 at day
3 of life (Fig. 6A),
returning to control values by day 6. Hyperoxia induces a
significant increase of about twofold in PCNA expression in neonatal
AEC2 by day 3 (Fig. 6B), suggesting that cell
proliferation and/or DNA repair are occurring. TGF-
activity and
PCNA expression were similar in control and hyperoxia AEC2 after the
removal of oxygen at day 10 (data not shown). The temporary
proliferative response of the neonatal AEC2, coincident with reduced
autocrine TGF-
activity, mimics the response to hyperoxia seen in
adult AEC2 (6).
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Dynamic activity of lung uPA during early neonatal growth is
disrupted by hyperoxia.
To determine the role of plasmin in neonatal lung TGF- activation,
we measured uPA in conditioned medium from control vs. hyperoxic lungs
of various ages, using a chromogenic assay, which detects colored
product released by cleavage of substrate by active uPA. uPA activity
is induced in the neonatal lung in the first week of life (Fig.
7), and this developmental response is
blunted by hyperoxia. Early in life, by day 3, hyperoxic
lungs have 1.5-fold higher uPA activity than control lungs, but by
day 8 hyperoxic lung uPA activity is ~30% lower than
normoxic lung activity and remains lower after the removal of the
oxygen. The developmental increase seen in uPA activity in normoxic
lungs parallels the TGF-
activity profile, suggesting a possible
role for plasmin in the activation of TGF-
during normal neonatal
lung alveolarization. However, no such correlation is seen in hyperoxic
lungs, suggesting that TGF-
activation may be mediated by another
protease during hyperoxia.
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Neonatal AEC2 from hyperoxic rat pups secrete significantly less
active uPA early in life than control neonatal AEC2.
uPA activity in conditioned medium from neonatal AEC2 isolated from
control vs. hyperoxic rat pups at days 3, 6, and
10 was compared (Fig. 8). A
2.5-fold decrease in uPA activity is seen in AEC2 cultured from
hyperoxic lungs compared with control AEC2. These data reflect the
active TGF- profile seen in neonatal AEC2 (Fig. 6). There was no
difference in active uPA levels between control and hyperoxic groups at
days 6 and 10.
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DISCUSSION |
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Treatments to improve systemic oxygenation and promote lung
maturity in premature infants can also result in permanent lung damage.
Impaired alveolar development is a uniquely neonatal response to
hyperoxic therapy (33). In this study, we examine the
temporal activity of three mediators of tissue remodeling during the
critical postnatal period, when the developing lung is most susceptible to hyperoxic insult. Activities of TGF- and candidate activators, gelatinases and uPA, were studied in whole lung and in cultured neonatal AEC2 during normal and hyperoxic alveolarization. All three
are known to be affected by hyperoxia (21, 30, 36).
The adult AEC2 remains relatively quiescent during normoxia but
proliferates under conditions of hyperoxia (31). Given the caveat that the response of a cell in vitro may differ from that in
vivo, autocrine TGF- activity differs among cultured fetal, neonatal, and adult AEC2. Cultured AEC2 isolated from normal adult rat
lungs, which are relatively quiescent, secrete higher levels of active
TGF-
[997 ± 54 pg · 106
cells
1 · 24 h
1 (6)]
than actively growing fetal AEC [757 ± 88 pg · 106 cells
1 · 24 h
1 (S. Buckley, unpublished data)]. In this paper, we
report that neonatal cultured AEC2 at 3-6 days of life secrete
1,488 ± 529 pg active TGF-
· 106
cells
1 · 24 h
1 (79-93% of
which is TGF-
3). In contrast, neonatal AEC2 cultured from
day 3 hyperoxic pups secrete less active TGF-
than
control neonatal AEC, while at the same time, increased TGF-
activity is recovered from the lung lavage. This apparent paradox of
decreased TGF-
activity secreted by hyperoxic AEC with increased
TGF-
activity recovered from the lavage is also seen in our adult
model of hyperoxia (6), suggesting that AEC2 in the
alveolus may create a local microenvironment different from that of the
rest of the lung. We speculate that the decrease in pericellular
TGF-
activity by AEC2 facilitates proliferation and repair.
Increased PCNA expression, seen in day 3 hyperoxic AEC2
compared with control AEC2, is consistent with this speculation.
Although the mechanism of activation of AEC-associated TGF-
is
unknown, the protease plasmin is a prime candidate (26).
Thus it has been postulated that AEC2 actively regulate plasmin
generation in the normal as well as injured alveolus (19).
Hyperoxic neonatal AEC2 secrete less active TGF-
and less uPA. The
decrease seen in AEC-associated uPA activity after hyperoxia may
reflect a shift in the balance between uPA and PAI-1, mediated by
increased oxygen tension, as found in retinal epithelial cells
(15). Activation of neonatal AEC2-associated TGF-
by
gelatinase is also a possibility, since gelatinase activity is also
decreased in hyperoxic neonatal AEC. Other possible activators of
TGF-
include metalloproteinases other than gelatinases and TNF-
(34)
TGF- activation in the whole lung shows a developmental profile. An
increase in activity is seen in hyperoxic lung by day 4,
which parallels then exceeds levels in the control lung. A similar
developmental increase is also seen in active TGF-
recovered from
lung lavages. Increased active TGF-
was recovered from the lavage of
hyperoxic lung, compared with control lung, as seen in our adult model
of hyperoxia (6), with the caveat that parenchymal leakage
may accompany the lavage of damaged lungs. Parallel increases in active
TGF-
in lung and lavage fluid suggest that macrophages and/or
infiltrating cells may be a major source of active TGF-
in the
neonatal lung after hyperoxia. Our previous studies in the
adult rat model of hyperoxia show a tight correlation between active
TGF-
recovered from macrophages and active TGF-
in the lavage
fluid (6). However, since we do not know the half-life of
active TGF-
in the lavage fluid, we cannot rule out AEC2-derived active TGF-
, secreted early in the injury process.
Our observation that MMP-9 activity parallels TGF- activity after
hyperoxia suggests a possible mechanism for TGF-
activation in
hyperoxic lung. TNF-
, also induced after neonatal hyperoxia (5), may also be activating TGF-
(34). It
is unlikely that uPA is activating TGF-
in hyperoxic neonatal lung,
because uPA activity does not parallel TGF-
activity in hyperoxic
lung (although it correlates well with active TGF-
in control lung).
Metalloproteinase activity regulates the development of all organs through ECM remodeling (1, 9, 27). In the rabbit neonatal lung, increased gelatinase expression and activity are seen during the alveolar phase of lung development (16). Although dynamic gelatinase activity is evident during alveolarization, individually, MMP-9 and MMP-2 are not essential, since MMP-9 and MMP-2 knockout mice have apparently normal lung phenotypes (20, 32).
Increased gelatinase activity is seen in neonatal hyperoxic lungs, and
several possible modes of activation are suggested by our findings. uPA
is a possible candidate, since uPA activity is concurrently induced by
hyperoxia. TGF- directs pancreatic morphogenesis through
metalloproteinase activation (27), and a similar process
may occur in the developing lung. MT-1-MMP may also be activating
gelatinase, since it is expressed at higher levels in the hyperoxic
neonatal lung. The decline of hyperoxia-induced gelatinase activity
during the recovery period may be mediated through TIMP-1.
Given the caveat that cells in vitro may respond differently from in
vivo, day 3 neonatal AEC2 from hyperoxic pups secrete less
active gelatinase than AEC2 from control pups. They also secrete less
uPA and less active TGF-, suggesting two possible mechanisms for
decreased gelatinase activity in AEC2. The decreased secretion of
active gelatinase seen in hyperoxic vs. control AEC2 differs from adult
AEC2, which secrete increased active gelatinases after hyperoxia
(7). It is possible that we have missed a very early
increase in activity, or alternatively, the hyperoxic response of
neonatal AEC2, with respect to gelatinase activation, may be intrinsically different from that of the adult AEC2, since it occurs
against a background of developmental increases in gelatinase activity.
Using the information obtained in this study, together with data from
our previous studies in hyperoxic models, we speculate that the initial
response of the lung to hyperoxia is an increase in TNF-, and
possibly other proinflammatory cytokines, in the exposed cells of the
alveolus (5, 12) followed by induction of TGF-
and MMP
activities in the alveolar fluid and lung parenchyma. Hyperoxic
induction of lung MMP suggests that administration of MMP inhibitors
with oxygen therapy could possibly minimize lung infiltration and
damage. When coadministered with bleomycin, the MMP inhibitor
Batimastat effectively reduced MMP-2 and MMP-9 activity in lung,
minimizing the characteristic fibrotic lesions, while reducing the
influx of macrophages and lymphocytes into the bronchoalveolar lavage
(BAL) (11). TGF-
had previously been considered the prime candidate for mediating bleomycin-induced fibrosis. However, our
data, showing induction of gelatinase activity during normal lung
alveolarization, argue against the use of MMP inhibitors during the
first week of neonatal life.
Thirty percent of the hyperoxic rat pups died on or after day
6, when lung TGF- activity peaks. We speculate that it is the sustained excess in TGF-
signaling that plays a key role in the subsequent alveolar hypoplasia and fibrosis seen in the survivors. This speculation is based on the comparable alveolar hypoplasia we
observed after adenoviral overexpression of active TGF-
in day
1 rat neonates (17). An important caveat is that both
of our studies by necessity involve rodent models and may not truly reflect events in human neonatal lung. However, in both the neonatal rodent model of hyperoxia and adenoviral overexpression of TGF-
1, the hyperoxic lesions are indeed anatomically and functionally similar
to the human (17, 33). Furthermore, we have shown that
TGF-
activity levels in the BAL from premature human infants correlate inversely with prognosis (24). Our new data,
showing induction of TGF-
activity in the normal lung in the first
week of life, suggest that treatment with agents directed against
hyperoxia-induced TGF-
activation should be coadministered with
oxygen therapy, as they would be delivered against a developmental
background of TGF-
activation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Warburton, Surgical Research, MS 35, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 17, 2002;10.1152/ajplung.00415.2001
Received 23 October 2001; accepted in final form 10 May 2002.
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