Induced p21Cip1 in premature baboons with CLD: implications for alveolar hypoplasia

Michael A. O'Reilly, Richard H. Watkins, Rhonda J. Staversky, and William M. Maniscalco

Department of Pediatrics, School of Medicine and Dentistry, The University of Rochester, Rochester New York 14642

Submitted 29 May 2003 ; accepted in final form 12 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells. The percentage of cells expressing p21 increased threefold in all PRN-treated animals, but different cell populations expressed it during disease progression. Between 2 and 6 days of treatment, p21 was detected in 30-40% of pro-SP-B cells. In contrast, only 12% of pro-SP-B cells expressed p21 by 14 and 21 days of treatment, by which time p21 was also detected in mesenchymal cells. Even though increased epithelial and mesenchymal cell proliferation occurs during disease progression, those cells expressing p21 did not also express the proliferative marker Ki67. Thus two populations of epithelial and mesenchymal cells can be identified that are either expressing Ki67 and proliferating or expressing p21 and not proliferating. These data suggest that p21 may play a role in disorganized proliferation and alveolar hypoplasia seen in newborn chronic lung disease.

bronchopulmonary dysplasia; cell proliferation; development; epithelium; oxygen; chronic lung disease


NORMAL LUNG DEVELOPMENT requires coordinated proliferation and differentiation of epithelial and mesenchymal cells. The observation that distal mesenchyme can promote branching morphogenesis and alveolar epithelial cell development when transferred to denuded tracheal endoderm is an elegant example of how distinct cell populations participate in this complex process (32, 36). Injury to the developing lung alters normal patterns of cell proliferation and may contribute to the disrupted tissue architecture typically seen in premature infants with chronic lung disease (CLD). Bronchopulmonary dysplasia (BPD) is the most extreme form of newborn CLD and was originally characterized as injury caused by oxygen and ventilation (22). The pathology findings include poor alveolization with severe inflammation and interstitial fibrosis. Through the use of exogenous surfactant and lower ventilatory pressures, a new and milder form of BPD has emerged, particularly in extremely premature infants (5). The lungs of these infants are less vascularized, and alveoli are fewer and larger, consistent with arrested alveolization (12). The observation that plasma obtained from premature infants can oxidize lipids suggests that their lungs might be oxidatively stressed, even though exogenous surfactant has reduced the need for high oxygen (19). Similarly, oxidized proteins have been detected in lavage obtained from infants with more severe lung injury (7). These findings suggest that enhanced oxidation of extremely premature infants may contribute to CLD.

The pathogenesis of BPD has been studied in a number of animal models. Perhaps the best animal model of human BPD is one in which premature baboons are delivered at 125 days (term is 185 days) and immediately resuscitated with artificial surfactant, positive pressure ventilation, and PRN oxygen for up to 3 wk (2). PRN or pro re nata refers to treatment "as the occasion arises." Because these animals are not exposed to high oxygen (typically <=0.5 FIO2) (16), they develop the milder form of BPD. Consistent with impaired alveolization, we recently documented type II cell hyperplasia and disrupted vascular development in these extremely premature animals (16, 17). Epithelial cell proliferation increased fourfold in 6- and 14-day PRN animals before decreasing by 21 days at which time pro-surfactant protein (SP)-B-positive epithelial cells lined hypoplastic alveoli and increased proliferation of interstitial cells was detected (16). Although it remains unclear what stimulates cell proliferation, both growth stimulatory and inhibitory pathways are likely to be activated at the same time, because proliferating and nonproliferating epithelial or mesenchymal cells lie adjacent to one another. Such an imbalance may disrupt interactions between epithelium and mesenchyme required for normal alveolization. Indeed, animals with CLD exhibited decreased expression of the angiogenic factor VEGF and its receptor Flt-1, along with dysmorphic capillaries that failed to develop subadjacent to the alveolar epithelium (17). Thus deregulated proliferation in BPD may result in accelerated growth of some cells that lie next to other cells whose growth is retarded. Molecular mechanisms underlying such disorganized alveolar cell proliferation and differentiation remains unknown.

Recent studies show that hyperoxia-induced oxidative stress can inhibit proliferation through increased expression of the cyclin-dependent kinase inhibitor p21Cip1/WAF1/Sdi1 (hereafter p21). This cell cycle inhibitor delays proliferation in G1 by blocking the G1- and S-phase cyclin-dependent kinases and by blocking association of DNA polymerases with the auxiliary protein proliferating cell nuclear antigen (14, 30). Hyperoxia inhibited growth of SV40-transformed rat type II epithelial cells and inhibited G1 cyclin E-dependent kinase activity through induction of p21 (3). Additional studies using flow cytometry confirmed that p21 exerts G1 arrest during hyperoxia (10, 29, 33). In adult mice, hyperoxia increased p21 mRNA and protein in airway epithelium and throughout the parenchyma (23). Consistent with the role of p21 to prevent cells from entering S phase, hyperoxia did not inhibit DNA replication in p21-deficient mice (24). Because p21-deficient animals exhibited continued DNA strand breaks, cell necrosis, inflammation, and myofibroblast proliferation during recovery in room air, p21 may also participate in tissue remodeling (34). In this capacity, p21 might prevent injured cells from replicating damaged DNA, thereby promoting the normal balance of type I and II epithelial cells and/or epithelial and mesenchymal cells required to restore normal lung architecture. Although p21 is not abundantly expressed in newborn mice, it is highly induced by hyperoxia (18). Because hyperoxia induces BPD-like pathology in newborn mice, this aberrant induction of p21 may be responsible for disrupting coordinated cell proliferation and alveolization of the developing lung. Together, these data suggest induced expression of p21 or other G1 kinase inhibitors in specific cell populations might lead to alveolar hypoplasia and premature lung development or abnormal remodeling of an injured lung.

Accumulating evidence suggests that disrupted cell proliferation contributes to CLD of the newborn. Although better interventions have reduced the clinical use of high oxygen concentrations, even low levels in premature infants may be hyperoxic compared with their normal in utero environment. Because hyperoxia induces p21, which inhibits cell proliferation, the current study investigates p21 expression in extremely premature baboons that develop lung disease.


    METHODS
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 DISCUSSION
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Animal exposures and tissue preparation. The Southwest Foundation for Biomedical Research (San Antonio, TX) provided lung samples from baboons delivered by elective hysterotomy at 125, 140, 146, 160, and 175 days' gestation, where term is 185 days. All animal procedures were reviewed and approved by the Animal Association for Accreditation of Laboratory Animal Care guidelines. Some animals delivered at 125 days' gestation were maintained with ventilator support and oxygen as needed to achieve normal blood-gas measurements for 2, 6, 14, and 21 days (2dPRN, 6dPRN, 14dPRN, or 21dPRN, respectively) (2, 37). At necropsy, the right lower lobe was removed, fixed in phosphate-buffered 4% paraformaldehyde, embedded in paraffin, and cut into 4-µm sections. Additional lobes were flash frozen in liquid nitrogen before being stored at -80°C.

Western blot analysis. Proteins (100 µg) were isolated from fresh frozen tissue, separated on SDS-PAGE, and transferred to polyvinylpyrolidine fluoride membranes as described (17). The membranes were blocked in 1x PBS containing 5% nonfat dry milk, followed by incubation in 1:500 mouse monoclonal anti-human p21, clone SX118 (BD PharMingen, San Diego, CA). Nonspecific interactions were removed by washing in phosphate-buffered saline with 0.05% Tween 20 (PBST), followed by incubation with goat anti-mouse secondary antibody (1:4,000; Southern Biotechnology, Birmingham, AL). The blots were washed again in PBST, and the conjugates were visualized by chemiluminescence (Amersham, Arlington Heights, IL). The blots were reprobed with anti-{beta}-actin antibody (1:5,000; Sigma, St. Louis, MO) as a loading control.

Immunohistochemistry. Primary antibodies used were mouse monoclonal anti-human Ki67, clone KiS5 (Dako, Carpinteria, CA), mouse monoclonal anti-human p21, clone SX118 (BD PharMingen), goat anti-human p21 (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-human pro-SP-B (Chemicon International, Temecula, CA).

After removal of paraffin, sections were rehydrated through graded ethanol and rinsed in deionized water for 5 min. Antigen retrieval was performed by heating the slides to simmer in 50 mM Tris, pH 9.5, for 15 min in a microwave oven, followed by cooling at 25°C for 20 min. Slides were rinsed in 50 mM Tris and 150 mM NaCl, pH 7.5 (TBS), and treated with 1% hydrogen peroxide in TBS for 20 min. Slides were rinsed in TBS and then in 50 mM Tris, pH 6, 0.05% Tween 20 (TBT6) and then blocked with 5% horse serum in TBT6. For tissues to be stained with 3,3'-diaminobenzidine (DAB), slides were incubated for 3 h in p21 (SX118) 1:50 in TBT6 plus 2% horse serum, rinsed 2 x 4 min in TBS plus 0.05% Tween 20 (TBST), incubated 45 min with biotinylated horse anti-mouse IgG 1:200, rinsed, treated for 30 min with Vectastain ABC Elite (Vector Labs, Burlingame, CA), rinsed, and stained with DAB substrate for 7 min. The slides were counterstained with methyl green.

For fluorescent staining, slides were blocked with 5% donkey serum in TBT6, and endogenous biotin was blocked with avidin/biotin blocking reagents (Vector Labs). Slides were incubated overnight at 4°C in p21 (C-19) 1:10,000 in TBT6 plus 3% donkey serum, rinsed 4 min in TBT6 and 2 x 4 min in 50 mM Tris-300 mM NaCl-0.1% Tween, pH 7.5. The tissues were then incubated 1 h with biotinylated horse anti-goat IgG 1:500 in TBST plus 3% donkey serum, rinsed in TBST, incubated in streptavidin-horseradish peroxidase 1:100 in TBST plus 5% donkey serum for 30 min, rinsed, and treated with biotinylated tyramide (Perkin Elmer, Torrance, CA) for 9 min, rinsed, incubated in streptavidin-Texas red 1:500 in TBST plus 5% donkey serum for 30 min, and rinsed. A second primary antibody of either mouse anti-Ki67 1:50 or rabbit anti-pro-SP-B 1:1,800 in TBT6 plus 3% donkey serum was then added for an overnight incubation at 4°C. After a rinse in TBST, donkey anti-mouse IgG-FITC or anti-rabbit IgG-FITC 1:200 in TBST plus 2% donkey serum was added for 1 h. Slides were rinsed, counterstained with 4',6-dia-midino-2-phenylindole (DAPI), and mounted using Vectashield (Vector Labs).

Quantitative immunohistochemistry. Random, noncontiguous fields of parenchyma were acquired using the x40 objective of a Nikon E-800 microscope and a SPOT RT camera. Five fields per lung were obtained from 2-4 separate animals at each gestational age or PRN treatment. Fields that contained a large airway or blood vessel were rejected. Different fluorescent filters were used to acquire images of each field displaying all nuclei (DAPI), p21 (Texas red), proliferating cells (FITC), or pro-SP-B (FITC). The images were merged to identify cells that were positive for Texas red or FITC. Quantification was performed using Metamorph (Universal Imaging, Downingtown, PA). A total of 131 fields containing 229-783 nuclei (429.5 ± 103.9, mean ± SD) were counted in this study. For each animal, the counts from all of the fields were summed and the following ratios determined: p21-positive/total cells; pro-SP-B-positive/total cells; p21- and pro-SP-B-positive/total cells; p21- and pro-SP-B-positive/pro-SP-B-positive; and p21- and pro-SP-B/p21-positive cells. The ratios for all animals at each time point were averaged and graphed as means ± SE.

Statistical analysis. The Kruskal-Wallis test was used to compare trends in PRN animals over time. The Wilcoxon rank-sum test was used to compare data between gestational control (GC) and PRN animals. Calculations were performed using Stata software with P < 0.05 considered significantly different.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
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P21 increases in lung development and BPD. Before determining whether p21 expression was altered by prematurity and BPD, we investigated its expression as lungs developed. P21 was faintly detected by Western blot analysis in 140-day gestational control (140dGC), 146dGC, 175dGC, and term animals, but not in 125dGC or adults (Fig. 1A). Expression was extremely low, and it was detected only when films were intentionally overexposed. These changes in expression were difficult to quantify because p21 was not detected at 125 days' gestation and its signal when detected was not much higher than background. This low level of expression, however, confirms other studies showing minimal p21 expression in newborn or adult mice exposed to room air (18, 23). In contrast, {beta}-actin levels did not change during development. Thus the low levels of p21 during development suggest that a small population of cells expressed it between 140 days' gestation and term.



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Fig. 1. P21 increases during lung development and chronic lung disease (CLD). Lung homogenates (100 µg) prepared from different gestational ages, term, and adult baboons (A) or from animals with CLD (B) were immunoblotted for p21. The blots were reprobed for {beta}-actin to ensure that comparable levels of protein were in all lanes. 125d, 125-day prematurely delivered baboon lungs; 14dPRN, animals treated pro re nata with O2 and ventilation for 14 days.

 

P21 expression in 125dGC, 140dGC, and 146dGC animals was then compared with that of animals born at 125 days and treated with PRN oxygen and ventilation for 14 and 21 days (Fig. 1B). Note that the appropriate age-matched controls for 14dPRN are 140dGC and for 21dPRN is 146dGC. At this level of film exposure, p21 was not detected in 125dGC, 140dGC, or 146dGC homogenates, whereas increased expression was detected in 14- and 21-day PRN-treated animals. {beta}-Actin levels remained constant. These findings reveal premature animals with CLD expressed more p21 compared with GC. Although p21 was difficult to detect, these findings prompted us to further investigate its expression by other methods.

P21 immunostaining in lung development and BPD. Immunohistochemistry (IHC) was used to identify cell-specific expression of p21 during fetal development and disease progression. Nuclear staining was detected in bronchiolar epithelial cells of 125dGC, 140dGC, and 146dGC animals (Fig. 2, A-C). P21 is trafficked to the nucleus by a nuclear localization signal in its carboxy terminus (14). Compared with 125dGC, staining appeared more intense in older animals. Less staining was observed in mesenchyme underlying airway, large blood vessels, or in alveolar cells. P21 continued to be expressed predominantly in airway epithelium of 160dGC and 175dGC animals, including term, but not in adults (data not shown). Overall these data suggest that the low level of p21 detected by Western blot analysis largely reflects expression in a small percentage of airway epithelial cells.



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Fig. 2. Cellular localization of p21 during normal lung development and CLD. P21 was immunostained in baboons delivered at 125dGC (125-day gestational control, A), 140dGC (B), 146dGC (C), baboons treated for 2 days PRN (2dPRN, D), 14dPRN (E), or 21dPRN (F). Compared with the GC animals, all animals with CLD had more staining in the parenchyma (arrows). Bar in F = 50 µm.

 

IHC was used to determine where p21 was expressed in PRN animals. Although p21 was not readily detected in parenchyma of 125dGC animals (Fig. 2A), it was readily detected in parenchyma of 2dPRN animals (Fig. 2D). Closer examination revealed p21 was predominantly expressed in cells lining the alveolar surface. A similar pattern of expression was observed in 6dPRN animals (data not shown). Although p21 was still expressed in parenchyma of 14dPRN and 21dPRN animals, it was detected in cells lining alveoli and within thickened alveolar septae (Fig. 2, E and F). Compared with the 140dGC or 146dGC animals, the 14dPRN and 21dPRN animals exhibited hyperinflated alveoli lacking secondary crests. Localized regions with the most abnormal histopathology in 21dPRN animals had the most p21 expression.

P21 expression in specific alveolar cells. We previously reported that alveolar expression of pro-SP-B, a gene expressed by epithelial cells, modestly increases during normal gestational development as overall cell proliferation decreases (16). In contrast, pro-SP-B expression rapidly increases within the first 6 days of PRN oxygen and remained elevated throughout disease progression. The proportion of pro-SP-B-positive cells that were proliferating increased from 3% in GC animals to 13% during the first 2 wk of treatment, after which time interstitial cell proliferation became predominant. These findings suggest that CLD is associated with markedly altered patterns of epithelial cell proliferation. To quantify and localize epithelial cells expressing p21, we used DAPI to mark all nuclei combined with double immunofluorescence for p21 and pro-SP-B. DAPI nuclei fluoresce blue, p21 expressing nuclei fluoresce red, and pro-SP-B cells fluoresce green (Fig. 3). The proportion of cells expressing each protein was quantified by image analysis. Between 125 and 146 days of gestation, the proportion of cells staining for p21 (red) or pro-SP-B (green) was low (Fig. 3, A and B). Approximately 5% of all parenchymal cells expressed p21, which remained constant over this time period (open bars, Fig. 4A). Similar to our previous study (16), the proportion of parenchymal cells expressing pro-SP-B modestly increased from 7.5 ± 0.6% of total cells in 125dGC animals to 9.3 ± 1.2% by 146dGC. Within this population, the proportion of pro-SP-B cells that also expressed p21 decreased from 3.9 ± 0.8% in 125dGC animals to 0.6 ± 0.4% in 146dGC animals (open bars, Fig. 4B). Likewise, the proportion of p21 cells that expressed pro-SP-B was extremely low (open bars, Fig. 4C). In fact, the total number of cells expressing both p21 and pro-SP-B in the entire parenchyma was <1% (open bars, Fig. 4D). This extremely low number of cells expressing p21 is consistent with our difficulty in detecting p21 by Western blot analysis. These data suggest accumulation of pro-SP-B-expressing cells during lung development is not associated with increased expression of p21.



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Fig. 3. P21 is expressed in pro-surfactant protein (SP)-B-positive epithelial cells. Sections prepared from 125dGC (A), 146dGC (B), 2dPRN (C), and 21dPRN (D) were immunostained for p21 and pro-SP-B. Immunofluorescence staining for p21 (Texas red, dashed arrows) and pro-SP-B (FITC, solid arrows) was combined with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent marker for double-stranded DNA (blue nuclei). Dual immunofluorescent cells containing p21 and pro-SP-B are denoted with yellow arrows. Bar in D = 50 µm.

 


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Fig. 4. Quantification of p21 and pro-SP-B-expressing cells during lung development and CLD. Total number of nuclei, number of nuclei containing p21, and number of pro-SP-B-positive cells were quantified by image analysis. Five random fields from 2-4 animals were analyzed. A: the proportion of cells expressing p21 significantly increased in CLD. Data are expressed as the percentage of total nuclei that are p21 positive. B: the proportion of pro-SP-B-positive cells also expressing p21 significantly increased in animals with CLD. C: the proportion of p21-positive cells also expressing pro-SP-B significantly increased in CLD. D: the proportion of all cells expressing both p21 and pro-SP-B was significantly higher in PRN-treated animals. Data in all graphs are means ± SE, *P < 0.05 compared with corresponding GC animals. Note that 6dPRN animals lack corresponding GCs, and 21dPRN values are obtained from 2 animals ({dagger}). The x-axes are not linear, and the scales of the y-axes are different.

 

A similar analysis was performed on PRN-treated animals (Fig. 3, C and D). Unlike GC animals, the proportion of cells expressing p21 or pro-SP-B markedly increased in the parenchyma of PRN-treated animals. Higher power resolution indicates that p21 was highly expressed in cells lining alveolar surfaces at early times with more interstitial staining as CLD developed. This apparent change in expression of p21 was confirmed by quantifying cells (Fig. 4). Compared with GC animals that expressed p21 in ~5% of all cells, PRN animals exhibited a two- to threefold increase in parenchymal cells expressing p21 (solid bars, Fig. 4A). The proportion of pro-SP-B cells that expressed p21 significantly increased 10-fold (solid bars, Fig. 4B). Although 40% of pro-SP-B cells expressed p21 during the first week of treatment, it sharply declined in 14dPRN and 21dPRN animals to 12.5%. This trend toward decreased p21 in pro-SP-B cells during 3 wk of CLD was statistically significant (P < 0.05). In contrast, the number of p21 cells that expressed pro-SP-B did not decline to the same degree (solid bars, Fig. 4C). A comparison of Fig. 4, A and D, shows that the total proportion of p21 expressing cells remains high in 14- and 21dPRN animals, whereas the proportion of cells expressing both p21 and pro-SP-B declines. All together, this suggests that dual expressing pro-SP-B and p21 cells rapidly appear within a few days of prematurity. As lung disease progresses, p21 is expressed by nonpro-SP-B interstitial cells, which is reflected by the increased staining within alveolar septae.

P21 is not expressed by proliferating cells. Our previous studies revealed that overall parenchymal cell proliferation declines during normal gestational development (16). This decrease in proliferation is disrupted by prematurity and is replaced by a rapid increase in proliferating pro-SP-B-positive cells followed by an increase in proliferating nonpro-SP-B-expressing interstitial cells. The observation that prematurity and BPD stimulate both proliferation and p21, a growth inhibitor, led us to investigate whether cells expressing p21 were proliferating. We used the nuclear protein Ki67 to mark proliferating cells because cells undergoing unscheduled DNA synthesis (DNA repair) do not express it. We previously showed that Ki67 immunostaining in premature baboons closely correlates with in situ hybridization of histone H3.2, an mRNA that is expressed during S phase of the cell cycle but not during DNA repair (16). Dual immunofluorescence staining for Ki67 and p21 in GC or 6dPRN- and 21dPRN-treated animals revealed that Ki67 was rarely detected in cells expressing p21 (Fig. 5). This figure is taken from a 21dPRN animal because p21 is expressed in both alveolar epithelial and interstitial cells by this time. In fact, dual p21/Ki67 cells were so rare that it was difficult to quantify. The finding that Ki67 was not detected in cells expressing p21 is consistent with p21 exerting cell cycle arrest.



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Fig. 5. P21 is not detected in cells expressing Ki67. Immunofluorescence staining for p21 (Texas red, dashed arrow) and Ki67 (FITC green, solid arrow) was combined with DAPI used to label all nuclei blue. Except for an occasional cell (yellow arrow), red p21 expressing cells do not overlap with green Ki67 proliferating cells.

 


    DISCUSSION
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 METHODS
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Supplemental oxygen, mechanical strain, and inflammatory mediators are the leading causes of CLD of the premature infant. Disorganized remodeling can occur when proliferation becomes altered as damaged cells fail to maintain normal rates of growth. Indeed, extremely premature baboons treated with oxygen and ventilation initially exhibit enhanced proliferation of epithelial cells that is followed by increased proliferation of interstitial cells (16). Although altered proliferation could be due to expression of growth stimulatory or inhibitory molecules, the current study provides evidence for increased expression of the growth inhibitory protein p21. Compared with GC, p21 rapidly accumulated in pro-SP-B expressing alveolar epithelial cells followed by expression in nonpro-SP-B-expressing interstitial cells. Cells expressing p21 lacked the proliferative marker Ki67, indicating that they were not dividing. These data suggest that abnormal expression of p21 might contribute to the substantially altered patterns of cell proliferation and accumulation that may lead to decreased alveolization, vascularization, and fibrosis seen in CLD of the newborn.

The animals used in this study were 125 days' gestation (comparable with 26 wk in humans). In this model, one of the earliest events observed during disease progression is the accumulation of pro-SP-B-expressing alveolar epithelial cells during the first 48 h following birth. Epithelial cell proliferation remained elevated until 14dPRN, at which time cuboidal type II cells lined the alveolar surface. Although increased proliferation of type II cells occurs, an important finding in the current study is that two populations of type II cells are observed. Approximately 12% of type II cells in 2dPRN animals are proliferating (16), whereas ~40% express p21 and are not proliferating (Fig. 4B, present study). Mesenchymal cells may also be distinguished during the latter stages of disease by their expression of p21 and rate of proliferation. Because of the thin squamous morphology of endothelial cells, identification of p21 in these cells will require dual fluorescent IHC with an endothelial marker, such as platelet endothelial cell adhesion molecule. We are developing tools to allow us to answer this question. Although unknown, it is unlikely that p21 is required for normal lung development because <5% of all cells express it. Aberrant expression of p21, however, may delay proliferating type II cells from exiting the cell cycle during their differentiation into type I cells. In this scenario, a nonproliferating population of type II cells would accumulate along the alveolar surface. Because type II cells can inhibit fibroblast proliferation (25), an overabundance might disrupt the complex interactions between epithelium and mesenchyme required for normal alveolization. An abnormal alveolar epithelium may also influence development of the microvasculature. Indeed, alveolar type II cells isolated from rabbits recovering from hyperoxia express VEGF, an angiogenic factor (15). Moreover, VEGF and its receptor Flt-1 decrease in premature baboons with CLD (17). Thus an imbalance in type II epithelial cell populations may profoundly affect mesenchymal cell proliferation and differentiation. This concept is consistent with our finding that abnormal epithelial cell proliferation and p21 induction precedes similar changes in the mesenchyme.

In addition to premature baboons, normal newborn rodents exposed to hyperoxia have been used to model BPD. Tritiated thymidine labeling studies in newborn rats exposed to room air revealed distinct growth patterns occur during postnatal lung development (13). The proliferative index of the vascular endothelium starts high at birth and decreases after 10 days. Likewise, fibroblast proliferation is high at birth and slowly subsides by 10 days. In contrast, type II epithelial cell proliferation starts low and markedly increases over 7 days before subsiding. By day 21 when lung development is complete, the labeling index of the whole lung approximates the very low levels seen in adults. As shown by tritiated thymidine or BrdU incorporation, these dynamic changes in cell proliferation and alveolization are disrupted when lungs are exposed to 100% oxygen (21, 35). Total cell proliferation is inhibited during the first 72 h of exposure followed by enhanced proliferation over the next 10-14 days that slowly subsides by 3-4 wk. Although growth inhibitory mechanisms remain unknown, p21 is a strong candidate because its expression increases in newborn mice exposed to hyperoxia (18). Additional studies using p21-deficient newborn mice should clarify how aberrant expression of p21 affects lung development.

Although mechanisms that stimulate p21 expression in CLD are unknown, it is now established that growth arrest occurs when cells are exposed to >40% oxygen, a level that damages DNA (31). In response to DNA damage, the tumor suppressor protein p53 accumulates in cells and exerts G1 growth arrest by transcriptionally increasing p21. Indeed, cultured epithelial cells exposed to hyperoxia exhibit DNA strand breaks, rapid accumulation of p53 and p21, and growth arrest in G1 (6, 28). Growth arrest is thought to protect cells by preventing replication of damaged DNA and allowing additional time for repair to occur. This is consistent with the observations that p21-deficient adult mice and cell lines are acutely sensitive to DNA damage caused by hyperoxia (10, 24). Indeed, enhanced survival and reduced DNA strand breaks are observed when oxygen-exposed cells are arrested in G1 by altering culture conditions (28). It is, therefore, intriguing to consider alveolar cells may express p21 in an effort to prevent replication of damaged DNA. It remains unclear, however, whether DNA is damaged in premature baboons that are exposed to an average maximal FIO2<=0.5, which is significantly less than the 0.85-1.00 typically used in postnatal animal models of hyperoxia (16).

In addition to p53, several other signal transduction pathways are known to stimulate p21 expression. The cytokine transforming growth factor (TGF)-{beta} can increase p21 transcription independently of p53 (26). Indeed, TGF-{beta} is abundantly expressed in the developing lung (27) and is induced by hyperoxia (1). Steroid hormones that influence lung maturation can also affect p21 expression. For example, retinoic acid prevents p21 induction and growth arrest of SV40-transformed type II cells, presumably by blocking TGF-{beta} signaling (20). P21 increased in newborn rats treated with dexamethasone, a synthetic glucocorticoid used to stimulate postnatal lung development (4). Although additional studies on p21 expression and function during CLD are needed, multiple signal transduction pathways known to regulate p21 expression become activated during disease progression.

The current study also showed that p21 was expressed in the conducting airway epithelium during normal lung development. In developing hamsters, p21 was detected in 13.5-day-gestation (term is 16 days), but not in 12.5-day-gestation, animals or adults (11). Although p21 was highly expressed in conducting airway epithelium, it was also detected in alveolar epithelium. As in the current study, p21 expression was inversely associated with Ki67 staining, consistent with its role in blocking cell proliferation. Its role in lung development remains unclear, however, because lung abnormalities are not observed in p21-deficient mice. Together, p21 appears to be a major inhibitor of cell proliferation during and following lung injury, but not necessarily during normal embryonic development.

This study has several limitations. One limitation of the study is defining where cells are in the cell cycle based solely on expression of Ki67. Although cells expressing p21 also lacked Ki67, consistent with exit from the cell cycle, they may actually be cycling slowly or be arrested in later phases of the cell cycle. Analysis of other cell cycle inhibitory proteins may clarify this issue. In addition to altering proliferation, as accumulating evidence suggests, it may also protect cells against apoptotic signaling (8, 9). It is conceivable that the aberrantly high levels of p21 contribute to CLD by preventing removal of excess cells. Although morphological signs of apoptosis were not readily evident in premature baboons, this remains to be further investigated. The effects of postnatal interventions, including steroids, surfactant, antibiotics, or nutrition on cell proliferation remain unknown. Although each PRN-treated animal did not receive exactly the same care, the changes in p21 expression within each treatment group were highly consistent. The small error bars in the graphs found in Fig. 4 attest to the high reproducibility of the data. Thus this model of CLD in premature baboons is highly reproducible and has many similarities with human BPD. Our findings, therefore, suggest that expression of p21 and changes in cell proliferation in human BPD should be investigated.

In summary, increased expression of the cell cycle inhibitor p21 was detected in alveoli of extremely premature baboons treated with supplemental oxygen and ventilation. P21 initially increased in pro-SP-B-positive epithelial cells before accumulating in mesenchymal cells. Cells expressing p21 rarely expressed the proliferative marker Ki67, indicating that they were not dividing. Thus two populations of epithelial and mesenchymal cells can be distinguished during disease progression: a smaller, highly proliferative population that lacks p21 and a larger, less proliferative population that expresses p21. These data suggest that aberrant expression of p21 during alveolization could contribute to the lung hypoplasia seen in BPD. A better understanding of p21 regulation and function during BPD might have high therapeutic potential.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-63400 (W. M. Maniscalco) and HL-58774 and HL-67392 (M. A. O'Reilly).


    ACKNOWLEDGMENTS
 
The authors thank Jacqueline Coalson, Bradley Yoder, and Vicki Winter for expertise with the premature baboon model.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. O'Reilly, Dept. of Pediatrics, Box 850, Univ. of Rochester, 601 Elm-wood Ave., Rochester, NY 14642 (E-mail: michael_oreilly{at}urmc.rochester.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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