Dynamics of metalloproteinase-2 and -9, TGF-beta , and uPA activities during normoxic vs. hyperoxic alveolarization

S. Buckley and D. Warburton

Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-beta , 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-beta , and uPA activities seen in neonatal lungs during the first days of life was significantly impacted by hyperoxia. In whole lung, gelatinase and TGF-beta 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-beta , 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-beta ; gelatinase A; gelatinase B; urokinase-type plasminogen activator; type II alveolar epithelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , 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)-beta is necessary for normal lung development, as suggested by the neonatal-lethal phenotype of TGF-beta 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-beta , metalloproteinases, and uPA. TGF-beta can be activated by metalloproteinases (35) and may, directly or indirectly, activate gelatinases (27). TGF-beta 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-beta 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-beta 3 (6), uPA (19), and gelatinases A and B (7, 30). The effects of hyperoxia on TGF-beta 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-beta , 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-beta , 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-beta , 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-beta , 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta , 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-beta activity. We measured TGF-beta 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-beta 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-beta 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-beta was corrected for cell number in the case of neonatal AEC2, or lung weight when lung homogenates were used.

TGF-beta isoform determination. Conditioned medium from AEC2 was incubated for 2 h at room temperature with saturating doses of neutralizing antibodies to TGF-beta 1 and TGF-beta 3 (R & D Systems, Minneapolis, MN), and the active TGF-beta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: a representative Western blot of neonatal lung lysates shows induction of gelatinases A and B [matrix metalloproteinase (MMP)-2 and MMP-9] and membrane-type (MT)-1-MMP expression in the first weeks of life and upregulation by hyperoxia. Three independent blots were scanned, and the bands were quantitated with Scion Image software. Hyperoxia results in significantly increased expression of MMP-9 on days 2, 4, and 6 (P < 0.01 for each), MMP-2 on days 2 and 4 (P < 0.0125 for each), and MT-1-MMP on days 8 (P < 0.05) and 10 (P < 0.025). In contrast, MMP-1 expression decreases after birth and remains unaffected by hyperoxia. B: a representative Western blot of neonatal lung lysates shows that tissue inhibitor of metalloproteinase (TIMP)-1 is barely detectable in control lysates but is induced by hyperoxia at day 2 with no decline over the first week. Scanning of independent blots showed significant induction in hyperoxic vs. control lungs (P < 0.0025, n = 3). In contrast, TIMP-2 is expressed from birth in control animals and is not induced by hyperoxia.

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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Fig. 2.   A: MMP-9 and MMP-2 activity was measured as color product proteolytically released by captured active MMP-9 or MMP-2. Data are presented as means ± SD (n = 3-4). Hyperoxia induces MMP-9 activity in neonatal lung by day 2 (hyperoxic vs. control *P < 0.05, day 2; **P < 0.025, day 4), which then falls to levels significantly lower than control by day 8 (hyperoxic vs. control *P < 0.05). B: hyperoxia induces lung MMP-2 activity by day 2 (hyperoxia vs. control, **P < 0.025) but falls to significantly less than control values by day 6 (*P < 0.05) through day 8 (**P < 0.025, n = 3).

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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Fig. 3.   A: an early response to hyperoxia at the epithelial cell level is reflected by decreased gelatinase activity. Conditioned medium from cultured neonatal type II alveolar epithelial cells (AEC2) from hyperoxic animals on day 3 of life has significantly decreased MMP-9 activity compared with medium from control AEC2 (***P < 0.0125, n = 3). By day 6, no differences are detected between control and "hyperoxic" cells. B: MMP-2 activity in the conditioned medium from neonatal AEC2 from hyperoxic animals is significantly less than control at day 3 (*****P < 0.005) and day 6 (**P < 0.025. n = 3).

Dynamic activity of TGF-beta in developing neonatal lung is disrupted by hyperoxia. The amount of active TGF-beta 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-beta 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-beta 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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Fig. 4.   Lungs were removed from hyperoxic and control rat pups at ages from newborn to 15 days of life, extracted for active transforming growth factor (TGF)-beta as described in METHODS, and assayed using the plasminogen activator inhibitor (PAI)-1 luciferase assay. Data are presented as means ± SD for the first 10 days of life and represent 3-6 animals for each time point from 4 individual hyperoxia exposures. Hyperoxic lungs have significantly less active TGF-beta at day 2 than control lungs (*****P < 0.005), but by day 6 the active TGF-beta in hyperoxic lungs significantly exceeds control levels (*P < 0.05) and remains significantly elevated compared with control lungs through day 10 (**P < 0.025). There were no differences detected between control and hyperoxic lungs after day 10 (data not shown).

Increased levels of active TGF-beta 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-beta (Fig. 5), which reflects whole lung TGF-beta (Fig. 4). There is sixfold more active TGF-beta 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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Fig. 5.   Lung washes from control and hyperoxic neonatal lungs from animals aged from birth to 13 days were assayed for active TGF-beta by the PAI-1-luciferase method. The data presented are as means ± SD; n = 3-4. A peak of TGF-beta activity occurs in lung washes from control neonates at 7 days of life. There is significantly more active TGF-beta recovered in the washes from hyperoxic rat pups at day 3 (**P < 0.025), day 6 (*****P < 0.005), and day 13 (*****P < 0.005) compared with controls. The lavage TGF-beta profile reflects the lung TGF-beta profile (see Fig. 4).

Neonatal AEC2 from hyperoxic rat pups secrete significantly less active TGF-beta 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-beta . 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-beta 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-beta 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-beta activity, mimics the response to hyperoxia seen in adult AEC2 (6).


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Fig. 6.   Neonatal AEC2 were isolated from control and hyperoxic pups by elastase digestion and IgG selection on days 3 and 6, when significant changes in active TGF-beta are seen in the whole lung and in the lavage fluids. Data represent means ± SD of 3 isolations per time point. A: active TGF-beta was measured in conditioned medium collected over 24 h from neonatal AEC2 using the PAI-1 luciferase assay. Neonatal AEC2 from hyperoxic lungs at day 3 of life have significantly less active TGF-beta than control AEC2 (**P < 0.025), but by day 6 the levels in control and hyperoxic conditioned medium are similar. B: Western analysis of lysates from control and hyperoxic neonatal AEC shows a significant induction of proliferating cell nuclear antigen (PCNA) in the "hyperoxic" AEC2 at day 3. This induction was shown to be 1.9 ± 0.24-fold over control values (P < 0.05, n = 3), as measured by scanning independent Western blots using Scion Image software. Induction of PCNA is indicative of DNA replication and/or repair and is concomitant with the downregulation of autocrine active TGF-beta seen in Fig 6A. C, control.

Dynamic activity of lung uPA during early neonatal growth is disrupted by hyperoxia. To determine the role of plasmin in neonatal lung TGF-beta 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-beta activity profile, suggesting a possible role for plasmin in the activation of TGF-beta during normal neonatal lung alveolarization. However, no such correlation is seen in hyperoxic lungs, suggesting that TGF-beta activation may be mediated by another protease during hyperoxia.


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Fig. 7.   Urokinase-type plasminogen activator (uPA) was measured in control and hyperoxic lung extracts using a chromogenic assay, which detects colored product released by cleavage of substrate by active uPA. Data are presented as means ± SD. Hyperoxic lung extracts contain significantly more active uPA than controls at day 3 (*P < 0.05, n = 4), but by day 8 controls have risen to levels significantly higher than hyperoxic (****P < 0.01, n = 4). In the first week of life, active uPA correlates with active TGF-beta in control lungs, but not hyperoxic lungs (Fig. 4), and correlates with both control and hyperoxic active lung MMP-9 activation (Fig. 2).

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-beta 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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Fig. 8.   Conditioned medium from control and hyperoxic neonatal AEC2 was assayed for uPA activity using a chromogenic assay. Data are presented as means ± SD. AEC2 from day 3 hyperoxic pups secrete significantly less active uPA than AEC2 from control pups (****P < 0.01, n = 3) and may explain the concomitant decrease in TGF-beta activation seen in Fig. 6.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta [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-beta · 106 cells-1 · 24 h-1 (79-93% of which is TGF-beta 3). In contrast, neonatal AEC2 cultured from day 3 hyperoxic pups secrete less active TGF-beta than control neonatal AEC, while at the same time, increased TGF-beta activity is recovered from the lung lavage. This apparent paradox of decreased TGF-beta activity secreted by hyperoxic AEC with increased TGF-beta 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-beta 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-beta 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-beta 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-beta by gelatinase is also a possibility, since gelatinase activity is also decreased in hyperoxic neonatal AEC. Other possible activators of TGF-beta include metalloproteinases other than gelatinases and TNF-alpha (34)

TGF-beta 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-beta recovered from lung lavages. Increased active TGF-beta 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-beta in lung and lavage fluid suggest that macrophages and/or infiltrating cells may be a major source of active TGF-beta in the neonatal lung after hyperoxia. Our previous studies in the adult rat model of hyperoxia show a tight correlation between active TGF-beta recovered from macrophages and active TGF-beta in the lavage fluid (6). However, since we do not know the half-life of active TGF-beta in the lavage fluid, we cannot rule out AEC2-derived active TGF-beta , secreted early in the injury process.

Our observation that MMP-9 activity parallels TGF-beta activity after hyperoxia suggests a possible mechanism for TGF-beta activation in hyperoxic lung. TNF-alpha , also induced after neonatal hyperoxia (5), may also be activating TGF-beta (34). It is unlikely that uPA is activating TGF-beta in hyperoxic neonatal lung, because uPA activity does not parallel TGF-beta activity in hyperoxic lung (although it correlates well with active TGF-beta 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-beta 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-beta , 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-alpha , and possibly other proinflammatory cytokines, in the exposed cells of the alveolus (5, 12) followed by induction of TGF-beta 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-beta 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-beta activity peaks. We speculate that it is the sustained excess in TGF-beta 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-beta 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-beta 1, the hyperoxic lesions are indeed anatomically and functionally similar to the human (17, 33). Furthermore, we have shown that TGF-beta activity levels in the BAL from premature human infants correlate inversely with prognosis (24). Our new data, showing induction of TGF-beta activity in the normal lung in the first week of life, suggest that treatment with agents directed against hyperoxia-induced TGF-beta activation should be coadministered with oxygen therapy, as they would be delivered against a developmental background of TGF-beta activation.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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