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Am J Physiol Lung Cell Mol Physiol 293: L151-L161, 2007. First published March 30, 2007; doi:10.1152/ajplung.00389.2006
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TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung

Hidehiko Nakanishi,1,3 Takahiro Sugiura,1,3 James B. Streisand,4 Scott M. Lonning,4 and Jesse D. Roberts, Jr1,2,3

1Department of Anesthesia and Critical Care and 2Division of Newborn Medicine in the Department of Pediatrics, and 3Cardiovascular Research Center, Massachusetts General Hospital, Boston, and Harvard Medical School, Cambridge; and 4Genzyme Corporation, Cambridge, Massachusetts

Submitted 3 October 2006 ; accepted in final form 21 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 DISCLOSURES
 REFERENCES
 
Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O2-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.

bronchopulmonary dysplasia; transforming growth factor-beta

BRONCHOPULMONARY DYSPLASIA (BPD) is a chronic lung disease in prematurely born infants, presumably caused by O2 toxicity and pressure trauma, that is an important cause of infant morbidity and mortality (21, 38). Patients who survive with BPD often have obstructive airway disease, pulmonary hypertension, and delay of growth and development. In the past, BPD was marked by inflammation, interstitial fibrosis, smooth muscle hyperplasia, and squamous metaplasia in the distal airways. However, recently introduced therapies have modified the influence of oxygen toxicity and mechanical trauma in premature lung injury and changed the pathophysiology of BPD. Now BPD is associated with disruption of the terminal stage of lung development. Microscopic inspection of the lungs of babies who have died from BPD reveals a partial to complete arrest in alveolar development (20). This results in a diminished number of alveoli and a reduction in gas exchange surface area in the lungs of babies with BPD. Moreover, the disruption of alveologenesis is associated with abnormalities in the assembly of the blood vessels in the distal lung, and in many infants with BPD, causes a dysmorphic pulmonary microvasculature (5, 9, 54). Similar abnormalities in the terminal stage of pulmonary development are observed in the injured lungs of full-term newly born mice and rats and in prematurely born baboons and lambs. Despite recent advances in the care of premature neonates, we find that BPD afflicts nearly one-third of infants that were born before 27 wk of gestation (29).

Transforming growth factor (TGF)-beta is an important mediator of the stimulatory and inhibitory cell development pathways that moderate normal early lung patterning (63). However, several recent studies suggest that excessive TGF-beta signaling adversely impacts alveologenesis during the later phases of pulmonary development and may play an important role in the etiology of newborn lung diseases. For example, in premature babies with lung injury, the level of TGF-beta in the bronchoalveolar lavage is increased and correlates with the severity of BPD (24, 28, 50). In addition, increased TGF-beta immunoreactivity has been observed in the peripheral areas of lungs of babies who have developed BPD (57). In preterm and adult animals with lung injury, the expression of TGF-beta and its receptors are upregulated (41, 66, 69). Others have shown that overexpression of TGF-beta in newborn mice and rats results in pulmonary changes that are similar to those observed in BPD (17, 61). Although these studies suggest that abnormal TGF-beta activity inhibits the terminal stage of pulmonary development, its role in disrupting alveologenesis and microvascular development in the injured newborn lung is unknown.

Based on these observations, we postulated that in the injured developing lung, excessive endogenous TGF-beta signaling might inhibit normal alveologenesis and vasculogenesis. Studies utilizing a newborn mouse lung injury model were performed to test the hypothesis that TGF-beta-neutralizing antibodies attenuate TGF-beta signaling and promote alveolar development. Chronic inhalation of 85% O2 was observed to increase TGF-beta signaling and decrease the maturation of the mouse pup lung. Importantly, preemptive treatment with an anti-TGF-beta antibody inhibited lung injury-induced TGF-beta signaling and promoted alveologenesis and normal pulmonary microvascular development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 DISCLOSURES
 REFERENCES
 
Experimental design. On embryonic days 17 and 19, time-dated pregnant C57BL/6 mice (Charles River) were treated with intraperitoneal injections of 0.5 ml of PBS without or with 10 mg/kg 1D11 (Genzyme), a pan-specific anti-TGF-beta IgG1 antibody, or MOPC21 (Sigma), an isotype-matched IgG. Within 12 h of birth, the pups of pairs of similarly treated mothers were pooled and then randomly divided into two litters that were continuously exposed, with their mothers, to either air or 85% oxygen for 10 days. Every 24 h, the mothers from paired litters were exchanged between the O2 exposure levels to diminish the effects of breathing high levels of O2 on them. The gas mixtures were blended using separately regulated and calibrated flow meters and medical grade O2 and N2 gases and introduced into a 680 L acrylic exposure chamber. An air lock permitted the exchange of the mothers and the freshening of food, water, and bedding without changing the pup oxygen exposure level. The fresh gas flow rate in the exposure chamber exceeded the calculated oxygen consumption of the mice within it by >10-fold (3). The oxygen level was measured by using paramagnetic methods (Servomex model 572). This exposure method produced a gaseous atmosphere with stable oxygen levels; the relative humidity and temperature in the exposure chamber did not exceed 60% and 70°C, respectively. All protocols were approved by the Subcommittee for Research Animal Studies of the Massachusetts General Hospital.

Tissue preparation. To obtain the lungs and hearts for analysis, pups were weighed and then killed with intraperitoneal injection of 200 mg/kg pentobarbital sodium. After a thoracotomy was made to permit the lungs to collapse, the trachea was cannulated with a 0.61-mm outer diameter polyethylene tube (PE10, Harvard Apparatus), the lungs were inflated with 3% paraformaldehyde and 0.1% glutaraldehyde in PBS at 22 cmH2O pressure for 30 min, and then the airway was ligated while the lungs were distended. The pup was then submerged in the fixative for 3 days at 4°C. Afterwards, the heart and lungs were dissected from the body and stored at 4°C in PBS for later processing and analysis. To obtain pulmonary tissue for frozen sectioning, after the pups were killed, the trachea was cannulated, the lungs were gently distended with Tissue Freezing Medium (TFM; Triangle Biochemical Sciences) and 20% sucrose in PBS (diluted 1:1 by vol), and the lungs were removed from the body. Subsequently, the lungs were covered with TFM and then frozen in isopentane kept on dry ice and stored at –80°C until use.

Detection of tissue TGF-beta. TGF-beta isoforms were detected in pup lungs using immunohistochemistry. Following citrate acid antigen retrieval, lung sections were blocked and exposed to rabbit anti-TGF-beta1 (Santa Cruz, sc-146), TGF-beta2 (sc-90), and TGF-beta3 (sc-82) antibodies or to preimmune rabbit serum (control). After extensive washing, the sections were exposed to biotinylated anti-rabbit antibody, avidin-biotin-peroxidase complexes (Vector Labs), and diaminobenzidine (DAB; Vector Labs) before being counterstained with hematoxylin.

Analysis of TGF-beta signaling. Nuclear phosphorylated Smad2 (p-Smad2) was detected in the lungs using immunohistochemistry. Frozen lung sections were fixed with 4% paraformaldehyde in PBS and permeabilized with 100% methanol. After blocking the sections with 5% goat serum in phosphate buffered saline containing 0.1% Tween 20 (vol/vol) (PBST), they were exposed to rabbit anti-p-Smad2 (Cell Signaling Technology) diluted in the blocking buffer overnight at 4°C. After washing the sections with PBST, they were incubated with a biotinylated anti-rabbit antibody, and, following PBST washes, exposed to avidin-biotin peroxidase complexes and then DAB before being counterstained with hematoxylin. p-Smad2 immunoreactivity was not detected in lung sections exposed to nonimmune rabbit serum instead of the anti-p-Smad2 antibody (data not shown). To quantify TGF-beta signaling, lung sections from a total of 12 pups that were treated with either PBS or 1D11 and exposed to either air or 85% O2 for 10 days, as described above, were stained for p-Smad2 as described above. From each section, five non-overlapping, 0.460-mm2 images of the lung periphery, which avoided the pleural surface and large airways and blood vessels, were obtained, and the DAB- and hematoxylin-stained nuclei were enumerated following color deconvolution (48), as implemented by Landini (26), using ImageJ (44). During the data acquisition and analysis, the investigators were not aware of the treatments that the lung specimens received.

Analysis of lung structure. The volume of the distention-fixed lungs was determined using the Archimedes principle. The effect of lung injury on alveolar development was objectively quantified by using stereological methods and by determining the mean linear intercept (Lm), the %air space volume density (%AVD), and the secondary septal density (32, 56, 65). The Lm is inversely proportional to the internal surface area of the lung (55); the %AVD inversely and secondary septal density directly correlates with the degree of alveolarization. Toluidine blue-stained sections of distention-fixed, methyl methacrylate-embedded left lung tissue were used for the structural studies. Using a systematic sampling approach described by Tschanz and Burri (58), four 0.60-mm2 section images from randomly oriented, transverse sections of the left lung that excluded large airway and vascular structures were obtained for the determination of Lm; within these fields, additional 0.15-mm2 section images were captured for the determination of %AVD and secondary septal density. Employing ImageJ (44) and a custom-written macro, the images were filtered and segmented using methods described by others (58). Lm was determined in the following manner. First, the images were inverted and then merged with one containing horizontal lines that were 90 µm apart using a bit-wise logical AND operation. After discarding chords <8 µm and >250 µm long, which are associated with pulmonary capillaries and conducting airways (53), the average chord length was then determined. The %AVD was determined using point counting methods detailed by Weibel (65). The median secondary septal density determined using four fields per animal was employed in the analysis. The obtainment and processing of the pulmonary image data occurred without knowledge of the treatment and exposure group of the lung specimens.

Lung staining and quantification of extracellular matrix proteins. The lung elastin and collagen assembly was determined using paraffin-embedded lung sections. Elastin staining was performed after the sections were treated with 0.5% potassium permanganate and 1% oxalic acid using Miller's elastin stain (34). Collagen staining was executed using 0.1% Sirius red in saturated picric acid (62). The %elastin volume density in the peripheral lung parenchyma (%EVD) was determined using a method similar to that described by Pierce and coworkers (42). Randomly oriented, non-overlapping 0.035-mm2 peripheral lung images were obtained in a similar manner as described above. The elastin-stained tissue was quantified following color deconvolution (26) using ImageJ. Following image thresholding, the number of pixels corresponding to elastin-stained and total parenchymal tissue was obtained, and the %EVD was determined as described by Pierce and coworkers (42). The median %EVD determined using four fields per animal was used in the analysis. The investigator capturing the lung images and processing the data was unaware of the treatment and exposure group of the lung specimens.

Evaluation of vascular development. Lectins from Griffonia simplicifolia, which have been observed to bind to endothelial cell {alpha}-D-galactosides (18, 33) and to preferentially identify the pulmonary microvasculature (23), were used to examine the effects of lung injury on pulmonary microvascular development. Paraffin-embedded lung sections were blocked and then incubated with a biotinylated IB4 isoform of G. simplicifolia lectin (Molecular Probes) overnight. After washing with Tris-buffered saline containing 0.1% Tween 20 (vol/vol), the bound lectin was detected using avidin-biotin-complexed alkaline phosphatase and Vector Red substrate (Vector Laboratories), and the sections were counterstained with hematoxylin. Immunohistochemistry was used to identify pulmonary smooth muscle cells (SMC) using paraffin-embedded lung sections, an anti-{alpha}-smooth muscle actin (anti-{alpha}-SMA) antibody (1A4; Sigma), and the method we previously described (46). To examine the specificity of these staining reactions, control sections were exposed to blocking solution and MOPC21, a nonspecific IgG, instead of IB4 lectin and 1A4, respectively. Right ventricular hypertrophy was assessed by determining the Fulton ratio (16). After the atria and large vessels were removed from the fixed pup hearts, the right ventricular free wall was dissected from the left ventricle and interventricular septum, and the right ventricle and left ventricle with septum were separately weighed using a protected scale.

The areal density of the pulmonary microvascular cells in the mouse pup lungs was analyzed using standard methods (65). Sections of lungs reacted with biotinylated lectin, or {alpha}-SMA antibody and Vector Red substrate, as described above, were used to detail the endothelial and SMC compartments of the microvasculature, respectively. Using the systematic sampling method detailed above, epifluorescent images were obtained of the peripheral lung avoiding the pleura and large airway and vascular structures. After using filtration and segmentation procedures, the %fluorescent volume density (%FVD) was determined by dividing the number of fluorescent pixels by the total number of pixels in the image. The median %FVD of three images per pup lung section was used in the analysis. During the obtainment and processing of the epifluorescent pulmonary image data, the investigators were unaware of the treatment and exposure group of the lung specimens.

Statistical methods. The effect of TGF-beta-neutralizing antibody treatment on p-Smad2 nuclear localization was assessed using chi-square analysis. The sample size for the stereological analysis was determined using methods described by Pocock (43). Preliminary studies indicated that 1D11 decreased the Lm of PBS-treated mice that breathed 85% O2 from 68.0 to 62.3 µm with a pooled SD of 4.2. Since these data indicated that 11.4 pup lungs per group would be required to demonstrate a salutary effect of 1D11 on Lm with a Type I error ({alpha}) of 0.05 and power of 0.90, 12 pup lungs were studied per treatment and exposure group. The lungs analyzed in the studies were obtained from at least four independent experiments. To detect a possible change in Lm by 10% by the isotypic IgG control antibody with an {alpha} of 0.05 and power of 0.9, six lungs were studied per group. The effects of TGF-beta-neutralizing antibodies on elastin, lectin, and {alpha}-SMA staining were assessed using a two-sided Student's t-test. The pup weights in paired litters were analyzed using a randomized complete block design (52). The data are presented as means ± SD and were compared using a factorial model of ANOVA. When significant differences were detected, a Scheffé test was used post hoc. Significance was determined at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 DISCLOSURES
 REFERENCES
 
Hyperoxic newborn lung injury is associated with increased p-Smad2 nuclear localization that can be modulated with anti-TGF-beta antibodies. Although the RNA levels of TGF-beta1 and TGF-beta2 are observed to increase in the postnatal rat lung (70), the expression pattern of the TGF-beta isoforms in the periphery of the developing lung is largely unknown. As shown in Fig. 1, a high level of TGF-beta1-3 immunoreactivity is detected in the epithelial cells of the proximal airways that extends to the level of the respiratory bronchioles, and in their associated pulmonary vessels. Importantly, TGF-beta1-3 immunoreactivity is also observed in the walls of the peripheral lung where alveolarization is diminished by lung injury. Because of these latter observations, an antibody that neutralizes the activity of TGF-beta1-3 in vitro (11, 49), 1D11, was used to examine the effects of TGF-beta modulation in the injured lung.


Figure 1
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  		Fig. 1. Transforming growth factor (TGF)-beta isoforms are expressed in the developing mouse pup lung. TGF-beta isoform immunoreactivity (brown) was detected in the bronchial epithelium (A, arrow), terminal and respiratory bronchioles (B; TB and RB, respectively), and in the terminal airways (C and D) of 10-day-old mouse pup lungs. There is abundant TGFbeta1-3 immunoreactivity in the walls of the terminal airways; counterstain was with hematoxylin. Closed scale bar, 50 µm; open scale bar, 100 µm; hatched scale bar, 30 µm.

 
Newborn pulmonary injury is associated with an increase in the abundance of TGF-beta (8, 24, 28). However, since TGF-beta is secreted in a latent form that can be bound to extracellular matrix ligands and thereby inactivated, it is not known if TGF-beta functional signaling increases in the injured newborn lung. Because increased TGF-beta signaling is associated with an increase in the phosphorylation and nuclear localization Smad2 or 3 (36), immunohistochemistry and nuclear p-Smad2 quantification were used to test whether hyperoxic pulmonary injury is associated with an increase in the nuclear compartmentalization of p-Smad2 in the mouse pup lung. With the use of this method, additional studies were performed to determine whether the increase in TGF-beta signaling could be modified by treatment with anti-TGF-beta antibodies. As shown in Fig. 2, compared with pup lungs treated with PBS and exposed to air, the level of nuclear p-Smad2 is increased in those treated with PBS and chronically exposed to 85% O2. Chronic inhalation of 85% O2 led to a 46% increase in the number of cells in the lung periphery with nuclear p-Smad2 immunoreactivity (cells with nuclear p-Smad2: PBS-air 41%, PBS-O2 60%; P < 0.05). Importantly, 1D11 treatment was associated with a decrease in p-Smad2 nuclear localization in the pup lungs exposed to high levels of oxygen; 49% of 1D11-treated O2-exposed lung cells had nuclear p-Smad2, which is different than that observed in PBS-treated lungs exposed to 85% O2 (P < 0.05). These observations indicate that hyperoxic lung injury increases TGF-beta signaling and that the excessive TGF-beta activity can be attenuated with TGF-beta-neutralizing antibodies. It is also interesting to note that some of the pulmonary cells in PBS-treated air-exposed lungs have nuclear p-Smad2. Moreover, treatment with 1D11 decreased the percentage of cells with nuclear p-Smad2 in the air-exposed lungs to 16% (P < 0.05). These data suggest that TGF-beta has some basal activity in the postnatal lung where it may play a role in regulating normal terminal lung development and that TGF-beta signaling is decreased in the developing lung by TGF-beta neutralization.


Figure 2
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  		Fig. 2. Treatment with anti-TGF-beta antibodies decreases TGF-beta signaling in the injured newborn lung. Immunohistochemistry was used to detect phosphorylated (p)-Smad2 (brown) in the lungs of 10-day-old pups treated as indicated. Compared with air-exposed lungs, those treated with PBS and exposed to 85% O2 for 10 days had more abundant p-Smad2 detected in the nuclei of distal airway cells (arrows indicate representative nuclear p-Smad2 immunoreactivity). In contrast, 1D11-treated, O2-exposed lungs had fewer nuclei with p-Smad2 reactivity than those treated with PBS and exposed to O2. The 1D11-treated, O2-exposed lungs also had a similar number of p-Smad2-stained nuclei as the lungs treated with PBS and 1D11 and exposed to air; these were counterstained with hematoxylin. Scale bar, 40 µm.

 
Alveologenesis is improved in the injured newborn lung by anti-TGF-beta antibody treatment. Chronic inhalation of high levels of oxygen by newborn rodents causes lung injury and inhibits terminal pulmonary development in a similar manner as observed in infants with BPD (7, 40, 64). Therefore, we tested whether treatment with TGF-beta-neutralizing antibodies improves alveolar development in the mouse pup lung under such conditions. Newborn mouse pups continuously breathed 85% O2 for 10 days because preliminary studies revealed that these exposure conditions diminished pulmonary alveologenesis while permitting a survival rate >80%. As shown in Fig. 3, chronic exposure to 85% O2 is associated with a decrease in terminal lung development. The distal airways of the injured pup lung exhibit a less complex interstitial structure and fewer secondary septae and alveoli compared with the lungs of air-breathing control pups. The apparent increase in distal air space area likely results from a decrease in interstitial tissue since the overall lung volume of the 85% O2-exposed lungs is not different from that of those exposed to air (lung volume, µl/g body wt: PBS and 85% O2 62 ± 8 vs. PBS and air 60 ± 5, P > 0.05). Importantly, exposure to anti-TGF-beta antibodies improved terminal pulmonary development in the injured newborn lung since it leads to a level of peripheral lung septation and air space area that is closer to that observed in the PBS-treated, air-exposed control lungs. In addition, treatment with 1D11 did not appear to be associated with lung toxicity. In both the 1D11-treated and air-exposed lungs, there was indistinguishable, minimal inflammatory cell infiltration in the distal airways. These observations suggest that moderation of TGF-beta signaling improves distal pulmonary development in the injured newborn mouse lung.


Figure 3
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  		Fig. 3. Anti-TGF-beta antibody exposure is associated with improved distal airway development in the injured newborn lung. Toluidine blue-stained sections of lungs of 10-day-old pups were treated as indicated. Treatment with PBS and exposure to 85% O2 was associated with increased distal air space area, and decreased numbers of secondary septae (arrows) were compared with lungs from air-exposed pups. Treatment with 1D11 improved distal airway development in O2-exposed pup lungs; there appeared to be more alveolar formation in these lungs. Scale bar, 200 µm.

 
To determine whether treatment with anti-TGF-beta antibodies improves alveologenesis in the injured newborn lung, the effect of 1D11 on objective, quantitative indexes of lung development was examined in pup lungs exposed to a high level of O2. As shown in Fig. 4, compared with exposure to air, breathing 85% O2 was associated with a 30% increase in Lm (PBS-air 50.7 ± 3.4 vs. PBS-O2 67.3 ± 3.7 µm), ~20% increase in %AVD (PBS-air 55.6 ± 3.7 vs. PBS-O2 67.8 ± 4.0%), and 76% decrease in secondary septal density (PBS-air 496 ± 7 vs. PBS-O2 119 ± 38 secondary septae/mm2). Importantly, compared with this change associated with PBS and O2 exposure, treatment of the O2-breathing lungs with 1D11 leads to a 30% decrease in Lm (1D11-O2 62.3 ± 4.4 µm), 51% decrease in %AVD (1D11-O2 61.6 ± 4.2%), and 38% increase in secondary septal density (1D11-O2 262 ± 50 secondary septae/mm2). These data suggest that TGF-beta importantly mediates the effect of injury on terminal lung development; neutralization of TGF-beta signaling with 1D11 improved lung surface area and distal air space area and enhanced the formation of distal airway structures that form alveoli. Interestingly, treatment with anti-TGF-beta antibodies alone was also associated with a modest decrease in %AVD. However, because 1D11 treatment did not change the Lm or secondary septal density in the air-exposed lung (P > 0.05), these data suggest that the level of TGF-beta signaling inhibition caused by 1D11 in this study might not affect a potential role of TGF-beta in regulating alveolar development. It is also possible that factors other than TGF-beta might have an important role in the terminal lung development in the lung.


Figure 4
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  		Fig. 4. In the injured lung, treatment with anti-TGF-beta antibodies improves quantitative indexes of alveolar development. In the periphery of PBS-treated mouse pup lungs, chronic exposure to 85% oxygen increased mean chord length (Lm; A) and the %air space volume density (%AVD; B) and decreased secondary septal density (C). Treatment with 1D11 ameliorated these changes in lungs chronically exposed to high levels of O2. *P < 0.05; n = number of secondary septae.

 
Additional studies using stereological methods were performed to investigate whether the protective effect of 1D11 could be due to its being an IgG1. Antenatal exposure to MOPC21, a 1D11 IgG isotype-matched control monoclonal antibody without a known antigen, did not alter alveologenesis, with no difference in Lm and %AVD in the pup lungs exposed to 85% O2 (Lm: MOPC21-O2 70.4 ± 7.1 µm vs. PBS-O2 68.0 ± 3.7 µm; %AVD MOPC21-O2 69.4 ± 2.6 vs. PBS-O2 68.9 ± 4.5%, both P > 0.05). These data are in agreement with those reported elsewhere (39).

Alveolar septal elastin organization is improved by anti-TGF-beta treatment in the injured newborn lung. Elastin is an important component of the alveolar wall, and its biosynthesis and extracellular assembly is critical for terminal lung development. In newborn mice with lung injury (60), as in the lungs of infants with BPD, the normal organization of elastin in the tips of the alveoli is disrupted and elastin is observed to reside in the walls of the distal airways. Because TGF-beta regulates extracellular matrix protein expression and assembly (27), the influence of TGF-beta-neutralizing antibodies on elastin biogenesis in the injured newborn lung was examined. Chronic exposure to high levels of O2 disrupted elastin assembly in the developing lung (Fig. 5A); elastin was observed to be disordered in the tips of the alveoli and spread throughout the septal wall. Furthermore, hyperoxic lung injury was associated with a 56% increase in %EVD in the peripheral lung parenchyma (Fig. 5B; P < 0.05). Importantly, exposure to TGF-beta-neutralizing antibodies improved the elastin deposition pattern in the oxygen-treated lungs. The elastin-staining pattern and the %EVD in 1D11-treated lungs exposed to 85% O2 is similar to that observed in those treated with PBS and exposed to air. Moreover, exposure to 1D11 is not associated with important changes in elastin in air-exposed pup lungs; the %EVD of PBS- and 1D11-treated lungs exposed to air was 11 ± 2% and 9 ± 1%, respectively (P = 0.18). These data suggest that excessive TGF-beta activity leads to the abnormal deposition of elastin in hyperoxic lung injury and that the protective effect of anti-TGF-beta antibodies on alveologenesis may be due, in part, to their improvement of extracellular elastin assembly. In agreement with others (7, 59, 64), no change in the collagen-staining pattern was observed during this early stage of newborn lung injury (data not shown).


Figure 5
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  		Fig. 5. Alveolar elastin deposition is improved in the injured newborn lung treated with anti-TGF-beta antibodies. A: Miller's elastin staining (black) of the peripheral lungs of 10-day-old mouse pups treated as indicated. High levels of elastin are expressed in the tips of secondary septa in the periphery of air-exposed pup lungs (arrows). In contrast, chronic exposure to 85% O2 is associated with an increase in elastin deposition in the walls of peripheral saccules and a disorganized expression pattern in the tips of many secondary septa (arrowhead). Treatment with anti-TGF-beta antibodies was associated with improved deposition of elastin in the tips of alveolar septae. Closed scale bar, 100 µm; open scale bar, 20 µm. B: the %elastin volume density (%EVD) in the peripheral lung parenchyma was determined by objective morphometric methods. Hyperoxic lung injury was associated with an increase in %EVD correlating with the increase in disorganized elastin deposition as depicted in A. Treatment with 1D11 led to improved elastin expression; the %EVD was not different than that measured in air-exposed pup lungs treated with PBS. There were 4 animals per group; *P < 0.05.

 
Treatment with an anti-TGF-beta antibody improves pulmonary microvascular development in the hyperoxic developing lung. In the developing mouse and rat, exposure to high levels of oxygen is associated with abnormal pulmonary microvascular development (10, 47). Since TGF-beta regulates the differentiation of cells that form blood vessels (45), we investigated whether modulation of abnormal TGF-beta signaling in the O2-injured newborn mouse lung with neutralizing antibodies attenuates dysmorphic microvascular development. Chronic exposure to 85% O2 was associated with abnormal pulmonary microvascular assembly; as shown in Fig. 6, the microvascular endothelial cell staining pattern is more anfractuous than that observed in the air-exposed lung. In many places in the peripheral airways, the lectin-staining pattern is arranged in two layers and suggests the existence of a double capillary network, which is observed in walls of distal airways of more immature lungs (2). Objective stereological quantification revealed that chronic exposure to 85% O2 was associated with an approximately fourfold increase in the volume density of lectin-stained cells in the periphery of the injured pup lungs (Fig. 7). Importantly, treatment with 1D11 improved the microvascular development in the injured lung. A more normal pattern of endothelial cells and a nearly 50% reduction in lectin %FVD were observed in lungs treated with 1D11 and 85% O2. Exposure to 1D11 was not associated with important changes in lectin reactivity in air-exposed pup lungs; the lectin %FVD of PBS- and 1D11-treated lungs exposed to air were 5.0 ± 1.7% and 4.2 ± 1.7%, respectively (P = 0.34). These data suggest that excessive TGF-beta importantly disrupts normal pulmonary microvascular development and that inhibition of abnormal TGF-beta signaling may improve pulmonary vascular assembly in the injured newborn lung.


Figure 6
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  		Fig. 6. Anti-TGF-beta antibodies improve pulmonary microvascular development in the injured developing lung. Griffonia simplicifolia isolectin B4 (lectin) staining of endothelial cell {alpha}-D-galactosidases and {alpha}-smooth muscle actin ({alpha}-SMA) staining of vascular smooth muscle cells in the periphery of 10-day-old pup lungs that were treated as indicated. The walls of lung septae exposed to 85% O2 are thickened and have an anfractuous pattern of lectin and decreased {alpha}-SMA staining. In contrast, the lungs treated with 1D11 and exposed to a high level of O2 have improved immunoreactivity for these vascular cell markers. Very little staining was observed in control lung sections (control) that were not exposed to lectin or to anti-{alpha}-SMA antibodies. Scale bar, 20 µm.

 

Figure 7
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  		Fig. 7. Quantification of the pulmonary microvasculature in the injured newborn mouse lung. A: pattern of indirect immunofluorescent staining of lectin-stained pulmonary microvascular endothelial cells and {alpha}-SMA-stained smooth muscle cells in the periphery of 10-day-old mouse pup lungs treated as indicated. In contrast with the PBS-treated and air-exposed lungs, those exposed to PBS and 85% O2 had increased lectin and decreased {alpha}-SMA staining. Treatment with 1D11 was associated with an improvement in the staining pattern of these microvascular markers in the O2-injured developing lung. Very little fluorescence was observed in control lung specimens (control) that were not exposed to lectin or to anti-{alpha}-SMA antibodies. Scale bar, 100 µm. B: the %fluorescent volume density (%FVD) of endothelial cells and smooth muscle cells in the peripheral lung. Injury of the developing lung was associated with an increase in lectin and decrease in {alpha}-SMA identification and %FVD. In contrast, exposure to TGF-beta-neutralizing antibodies improved the detection and volume density of these pulmonary microvascular cells markers. *P < 0.05; n = 7 each group.

 
Since SMC influence vascular development, we examined whether attenuation of abnormal TGF-beta signaling improves the abundance and distribution of vascular SMC in the injured newborn lung. Hyperoxic pulmonary injury was associated with a decrease in the abundance of SMC identified by {alpha}-SMA immunoreactivity in the distal lung (Fig. 6). The %FVD of {alpha}-SMA staining decreased by ~70% in the pup lungs chronically exposed to 85% O2 (Fig. 7). In contrast, treatment with anti-TGF-beta antibodies increased the amount of SMC observed in the injured lung. Exposure to 1D11 is not associated with important changes in {alpha}-SMA reactivity in air-exposed pup lungs; the {alpha}-SMA %FVD of PBS- and 1D11-treated lungs exposed to air were 14.2 ± 1.7% and 14.8 ± 1.3%, respectively (P = 0.65). These data suggest that TGF-beta neutralization has a salutary effect on the injured pulmonary microvasculature of the newborn. Since neomuscularization of peripheral pulmonary arteries, right ventricular hypertrophy, and pulmonary hypertension are not observed in early stages of hyperoxic newborn lung injury, these studies do not indicate whether anti-TGF-beta antibodies can prevent advanced pulmonary vascular disease in the injured neonatal lung. In this model, immunohistochemistry with {alpha}-SMA antibodies did not reveal an increase in pulmonary artery muscularization in pup lungs exposed to 85% O2 for 10 days (data not shown). In addition, the right ventricular mass of the pups breathing high levels of oxygen for this duration of time was not increased (mass RV:LV and septum, n = 14 each group: PBS-air 0.32 ± 0.04; PBS-85% O2 0.25 ± 0.05, P > 0.05).

Somatic growth and treatment with anti-TGF-beta antibodies. Somatic growth is often decreased in newborns with lung injury. Because treatment with anti-TGF-beta antibodies improved alveologenesis and vasculogenesis of the injured lung, we tested whether treatment with 1D11 enhances the growth of the hyperoxic pups. Following 10 days, the somatic weights of the 85% O2-breathing pups were nearly 30% less than what was observed in those that breathed air (body weight, grams, PBS-air 5.26 ± 1.18, PBS-85% O2 3.74 ± 1.0; n = 10–15, P < 0.05). Exposure to anti-TGF-beta antibodies improved the weight gain of pups exposed to high levels of O2; the weight of pups treated with 1D11 and exposed to 85% O2 was 4.62 ± 0.85 g and not different from the PBS- or 1D11-treated air-breathing controls.


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In the present study, TGF-beta was observed to directly modulate newborn lung injury. Continuous inhalation of a high level of O2 by newborn mice during the terminal stage of lung development increased TGF-beta signaling and severely decreased alveologenesis and disrupted vasculogenesis in the lung periphery. Importantly, exposure of the hyperoxic lung to an anti-TGF-beta antibody partially rescued this phenotype. Neutralization of abnormal TGF-beta signaling in the injured newborn lung improved pulmonary alveolar development, elastin deposition, and the microvascular structure and increased somatic growth. These observations suggest that TGF-beta-neutralizing therapies may have an important role in ameliorating the development of the injured newborn lung.

Recent indirect evidence suggests that TGF-beta modulates the terminal stage of development of the injured lung. This concept is supported by the identification of TGF-beta in the terminal airways, pulmonary effluents of premature babies with lung injury, and observations of the effects of TGF-beta in the developing lungs of mice and rats. For example, the level of TGF-beta1 observed in the tracheal effluents of preterm babies appears to be highest in those that develop BPD (24) and correlates with the severity of the illness (28). However, it is uncertain whether these increased levels of TGF-beta belie its importance in the pathogenesis of newborn lung disease or whether they result from the stimulation of TGF-beta expression by pathogenic cytokines and compounds or TGF-beta released by cells transiting the lung that directly modulate pulmonary development through other mechanisms. Moreover, it is unknown whether the increased pulmonary effluent TGF-beta levels are associated with an increase in TGF-beta signaling in the injured lung parenchyma. It is possible that downregulation of TGF-beta receptors and Smad proteins and increased expression of TGF-beta neutralizing proteins inhibit TGF-beta signaling in the injured lung. Moreover, although accumulating data indicate that TGF-beta regulates early events in fetal pulmonary development, such as branching morphogenesis (68), abnormalities in branching morphogenesis are not observed in the lungs of infants with BPD. However, studies suggest that excess TGF-beta during the period of terminal lung development can affect alveolarization. For example, infection of newborn rat lungs with an adenovirus that encodes TGF-beta1 has been observed to produce enlarged distal air spaces (17). In addition, overexpression of TGF-beta1 in the lung epithelium inhibits terminal pulmonary development in the newborn mouse (61). Recently, Neptune et al. (37) observed that mice deficient in fibrillin-1 have increased pulmonary TGF-beta signaling and decreased alveolarization. In this model of Marfan disease, distal airway development importantly improved following exposure to TGF-beta-neutralizing antibodies. However, it is uncertain whether the level and distribution of TGF-beta that was investigated in these studies replicate what occurs in the injured developing lung and thereby functionally inhibits terminal pulmonary development. In the studies described herein, hyperoxic pulmonary injury increased TGF-beta signaling in the newborn mouse lung. Chronic exposure to a level of oxygen that decreases alveolarization was noted to increase p-Smad2 nuclear localization, which is indicative of TGF-beta activation. Furthermore, pretreatment of the injured newborn lung with an anti-TGF-beta antibody not only attenuated pulmonary TGF-beta signaling but also promoted alveologenesis and improved the pattern of elastin deposition in the septal wall of newly formed alveoli. These observations suggest that endogenous TGF-beta has an important direct role in the inhibition of alveolarization that is observed in the injured developing lung.

Injury of the developing lung is associated with disruption of pulmonary microvascular development. Normally, the temporal and spatial development of the lung vasculature follows that of the airways (19). In the canalicular stage of lung development, small blood vessels coalesce around the developing future distal air spaces (13). During the saccular stage, a dual layer of capillaries is observed in the forming secondary septae. Subsequently, as the septae thin, the double capillary layer becomes a single one. Data suggest that injury of the lung during the canalicular and saccular stages of lung development causes the formation of dysmorphic blood vessels in the lung periphery. For example, the expression pattern of PECAM-1, a constituent protein of endothelial cells, has been observed to be abnormally distributed in the lung septal walls of infants who were born during this stage of lung development and had died from BPD (5, 12). The abnormal peripheral pulmonary vascular development results, in part, from alterations in the expression vascular growth factors (10). For example, in the injured developing lung of animals (30, 31) and infants with BPD (5), VEGF expression is diminished.

TGF-beta regulates the expression of several genes that modulate the differentiation, proliferation, and migration of cells that are required for normal pulmonary vascular assembly and maturation (for a review, see Ref. 54). Moreover, data indirectly suggest that excess TGF-beta during the terminal phase of lung development causes dysmorphic or imbalanced development of blood vessels in alveolar septae. For example, overexpression of TGF-beta1 in the lung epithelium has been reported to decrease PECAM-1 expression and septal pulmonary vessels in the lungs of developing mice (67). However, it is unknown whether endogenous TGF-beta has a direct role in disrupting alveolar vessel development in the injured newborn lung. We observed that chronic hyperoxia in the mouse pup is associated with an abnormal pattern of endothelial and SMC assembly into the pulmonary microvasculature. Although the tortuosity and volume density of endothelial cells in these vessels were increased in the hyperoxic pup lung, these qualities of the microvascular SMC were markedly decreased. Furthermore, because this abnormal microvascular patterning was improved with exposure to TGF-beta-neutralizing antibodies, these data suggest that TGF-beta has an important and direct role in mediating the vascular pathology of the developing lung.

Although short-term modulation of TGF-beta activity in newborns has not been tested clinically, several studies indicate that anti-TGF-beta antibodies do not cause adverse effects in mature animals (4, 15, 35). Moreover, although targeted disruption of TGF-beta2, TGF-beta3, TGF-beta receptor II, Smad2, and Smad4 in mice results in lethal defects (reviewed in Ref. 14), the scope and types of the malformations observed in these models suggest that TGF-beta's important influence in development occurs during early fetal development and may be completed before an anti-TGF-beta therapy for the prevention of BPD is contemplated. Early investigations revealed that mice harboring a targeted disruption of TGF-beta1 develop diffuse and lethal inflammation when they are ~3 wk old (25, 51). However, this likely reflects an effect of TGF-beta1 inhibition on early fetal immunoregulatory development since postnatal inhibition of TGF-beta1 with neutralizing antibodies does not cause inflammation in adult mice (49). Moreover, in the lungs of the anti-TGF-beta antibody-treated mouse pups reported herein, no important inflammation was observed, and the hyperoxic mice treated with 1D11 thrived compared with those treated with PBS. It is also possible that inhibiting TGF-beta activity will promote the formation of malignancies (6). However, it is unlikely that short-term inhibition of TGF-beta activity does this. Despite many animal studies of TGF-beta neutralization, no examples of malignant transformation have been reported. Importantly, the mouse pups exposed to the TGF-beta-neutralizing antibody in this study thrived; the growth of pups breathing air and treated with 1D11 was not different from those treated with PBS alone. Moreover, the growth of pups exposed to TGF-beta-neutralizing antibody and high levels of oxygen was improved compared with those treated with saline and oxygen. The effects of TGF-beta neutralization on normal pulmonary alveolar and microvascular development are incompletely understood. However, it is likely that the effects of altering TGF-beta signaling on pulmonary maturation depend on what is developing in the lung when the modulation of TGF-beta signaling occurs. For example, although studies of TGF-beta3 null mutant mice suggest that decreasing TGF-beta signaling in the embryonic and early fetal period inhibits the development of proximal airway structures and results in lungs with a pseudoglandular appearance (22), others suggest that short-term inhibition of TGF-beta near the time of birth does not affect the development of these structures and actually promotes alveolarization (37). Nevertheless, although we observed the 1D11 treatment decreases TGF-beta signaling and %AVD in the air-exposed pup lung, it did not alter the secondary septal density, Lm, lung volume, expression of elastin, and microvascular markers in the developing lung or affect pup growth. Additional studies of the long-term effect of TGF-beta neutralization on the terminal development of the lung will further inform the clinical use of this approach in the modulation of pulmonary injury.

Potential limitations of our investigation include the possibility that higher levels of anti-TGF-beta antibodies might have completely reversed the effect of hyperoxia on mouse lung development. However, the level of 1D11 used in our investigations was observed to importantly inhibit TGF-beta signaling in the hyperoxic pup lungs (Fig. 2). It is likely that some inhibition of terminal pulmonary development remains despite TGF-beta inhibition because other factors aside from TGF-beta mediate the effects of hyperoxic injury on lung structure. In addition, it is unknown whether anti-TGF-beta antibodies prevent the pulmonary fibrosis and vascular changes that are observed in other models of lung injury. In the model of lung injury employed herein, fibrosis, pulmonary neomuscularization, and right ventricular hypertrophy were not observed. However, in this respect, the model used herein closely replicates the balance of pathological findings observed in infants with BPD today. Nevertheless, since fibrosis and pulmonary remodeling can be observed in some models of lung injury, particularly during the late recovery phase of pulmonary disease, it might be important to investigate whether or not anti-TGF-beta therapies are protective in them.

In summary, an anti-TGF-beta therapy was observed for the first time to protect the injured newborn lung from inhibition of alveologenesis and vasculogenesis associated with exposure to high levels of O2. Additional studies may further delineate the protective mechanisms, safety, and the optimal dose and duration for use of this exciting new therapy.


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While the present work was being reviewed, Alejandre-Alcazar and coworkers (1) reported studies in which chronic exposure to high levels of oxygen was observed to increase the level of TGF-beta signaling in the injured mouse pup lung. These data were generated using whole lung samples and suggest that the overall increase in TGF-beta signaling might have a role in a global response of the lung to injury. Our results map increased TGF-beta activity to the periphery of the injured developing lung where some of the increased TGF-beta activity reported by Alejandre-Alcazar and coworkers might have special relevance to TGF-beta inhibition of pulmonary alveolar and microvascular development. Together, these observations support the important direct role of TGF-beta in newborn lung injury.


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H. Nakanishi and T. Sugiura received support from the Nitric Oxide Interest Group of Japan.


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This work was supported by a Genzyme-Partners Health Care Award for Translational Research.


   ACKNOWLEDGMENTS
 
We thank Rosemary C. Jones for advice on lung structural analysis, Göran Mattsson for information about lectin staining of the pulmonary microvasculature, Richard C. Gregory, Charlene Manning, and Bill Weber for help with the TGF-beta isoform immunolocalization in the lung, and Patricia K. Donahoe for thoughtful review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Roberts, Jr., Cardiovascular Research Center, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129 (e-mail: roberts{at}cvrc.mgh.harvard.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.


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