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Published ahead of print on July 10, 2003, doi:10.1165/rcmb.2003-0102OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 38-50, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0102OC

Abnormal Alveolar Development Associated with Elevated Adenine Nucleosides

Suman K. Banerjee, Hays W. J. Young, Andrea Barczak, David J. Erle and Michael R. Blackburn

Department of Biochemistry and Molecular Biology, University of Texas, Houston Medical School, Houston, Texas; and Department of Medicine, University of California, San Francisco, California

Address correspondence to: Dr. Michael R. Blackburn, Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail: Michael.R.Blackburn{at}uth.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenosine signaling has been characterized in various physiologic systems, but little is known about the role of adenosine signaling in lung development. Alveogenesis and microvascular maturation are the final stages in lung development in mammals. Alveogenesis in the mouse begins on Postnatal Day 5, when the process of secondary septation plays a pivotal role in the expansion of the alveolar sacs and microvascular maturation. Adenosine deaminase null mice (ADA-/-) exhibit abnormalities in alveogenesis in association with elevated lung adenosine levels. Large-scale gene expression analysis of ADA-/- lungs using oligonucleotide-based microarrays revealed novel relationships between gene expression patterns and elevated lung adenosine during the stages of alveolar maturation. Genes regulating apoptosis, proliferation, and vascular development were shown to be altered, and decreased cell proliferation in association with increased alveolar type II cell apoptosis was shown to contribute to abnormal secondary septation in these mice. ADA enzyme therapy allowed for normal patterns of apoptosis, proliferation, and alveolar development in association with prevention of adenosine elevations. These findings were correlated with the presence of adenosine receptors in the developing lung, suggesting the involvement of receptor signaling. These studies provide evidence that elevated lung adenosine can lead to abnormal alveogenesis by disrupting patterns of cell proliferation and apoptosis.

Abbreviations: adenosine deaminase–deficient, ADA-/- • bronchopulmonary dysplasia, BPD • bromodeoxyuridine, BrdU • complementary deoxyribonucleic acid, cDNA • immunohistochemistry, IHC • kinase insert domain protein receptor, KDR • polyethylene glycol modified-ADA, PEG-ADA • reverse transcription polymerase chain reaction, RT-PCR • sodium hydroxide, NAOH • significant analysis of microarrays, SAM • sodium dodecyl sulfate, SDS • surfactant proteins, SP • tissue transglutaminases, Ttg • terminal transferase-mediated dUTP nick end labeling, TUNEL • vascular endothelial growth factor D, VEGF-D


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammalian lung development is a complex morphogenetic process that begins around mid-gestation and continues through early postnatal life (reviewed in Ref. 1). Alveogenesis, the final maturation step in lung development, begins at Postnatal Day 4 in mice (2). The process involves the septation of alveolar saccules into mature alveoli to increase surface area and hence the oxygen exchange capacity of the lung (3). Cellular processes such as migration and proliferation play important roles in this septation process. Consistent with this, various growth factors and signaling molecules have been implicated in the process of alveolar septation (4, 5). Development of the lung microvasculature is also thought to play a key role in alveogenesis (6). However, a detailed understanding of the physiologic processes governing postnatal alveogenesis remains incompletely defined.

Adenosine is a signaling molecule that is produced constitutively by cells under normal conditions and can be produced in high concentrations at sites of tissue injury. Signaling through adenosine receptors can activate numerous cellular processes depending on the receptor subtype and the levels of available ligand (7). Adenosine signaling is largely thought to serve cytoprotective or anti-inflammatory roles (8); however, apoptotic (9) and proinflammatory functions (10) have been described. These heterogeneous effects are not surprising given the wide variety of signaling pathways accessed through adenosine receptor activation. Whereas adenosine signaling has been well characterized in physiologic systems and in response to acute injury or inflammation, little is known with regard to the role of adenosine signaling in lung development.

Recent findings using adenosine deaminase–deficient (ADA-/-) mice (11) suggest that the adenosine signaling pathway might play a role in alveogenesis (12). ADA plays a critical role in controlling the concentration of adenosine in cells and tissues, thereby affecting many areas of intercellular signaling (13). In the absence of ADA, the uncontrolled elevations of adenosine in vivo unleash a variety of signaling cascades, allowing one to analyze the phenotypic and metabolic consequences of ADA deficiency. When examined developmentally, it was found that adenosine levels were normal in the lung at birth, but increased significantly at Day 5 and then again at Day 10 in ADA-/- mice (14). When the lung structure of ADA-/- mice was examined, a severe defect in alveogenesis was observed between Days 5 and 10 (12). Treatment of these animals with polyethylene glycol–modified ADA (PEG-ADA) enzyme therapy prevented this alveolar defect in association with preventing elevations in lung adenine nucleosides early in life (12, 15). These studies suggested that elevated lung adenine nucleosides (adenosine, deoxyadenosine) could lead to abnormal alveogenesis. However, little to nothing is known with regard to the presence of adenosine receptors in the developing lung or the involvement of this signaling pathway in the processes that govern secondary septation or vascularization during alveogenesis. Deciphering the precise role of adenosine signaling in vivo remains a challenge as a result of complex dynamics of adenosine metabolism and the absence of adequate in vivo model systems. This makes the ADA-/- mouse an attractive in vivo model to study the specific roles of purinergic signaling in aspects of lung development and disease.

We examined alveogenesis in Postnatal Day 0, 5, and 10 control and ADA-/- mice, and in ADA-/- mice treated with ADA enzyme therapy to prevent accumulation of adenosine in the developing lung. Microarray analysis and clustering revealed distinct groups of differentially expressed genes on Day 5, the stage at which alveogenesis is normally initiated. Genes regulating apoptosis, proliferation, and vascular development in the alveoli were examined further, and our findings indicated that decreased cell proliferation in ADA-/- lungs in association with increased alveolar type II cell apoptosis on Day 5 contributed to abnormal secondary septation. We also demonstrate that regulation of apoptosis and cell proliferation by controlling lung adenosine levels with ADA enzyme therapy prevented abnormal secondary septation in ADA-/- lungs. The presence of adenosine receptors during these critical stages of lung development further implicated adenosine signaling in abnormal alveogenesis. Addressing the role of adenosine in alveogenesis is important from a clinical perspective in that lung hypoxia is a major source of adenosine production, and factors that induce lung hypoxia in neonates might lead to complications in normal alveogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic Mice and ADA Enzyme Therapy
ADA-/- mice were generated and genotyped as described previously (11). Control mice were either wild-type or heterozygous for the null Ada allele, as there was no phenotype seen in heterozygous animals (11). All mice were housed in cages equipped with micro-isolator lids and maintained under strict containment protocols. Effects of ADA enzyme therapy were monitored by injecting intramuscularly control or ADA-/- mice with dosages of PEG-ADA designed to deliver 100–500 U/kg body weight (12). Injections were given on Postnatal Days 1 and 4 before harvesting lungs on Day 5.

Tissue Specimens
Lungs were harvested from Postnatal Days 0, 5, and 10 ADA-/- mice and corresponding controls (n = 5 animals per group per time point). Lungs which were used for RNA isolation and nucleoside determinations were immediately snap-frozen in liquid nitrogen. Lungs which were used for histopathologic studies were fixed in 4% paraformaldehyde overnight at 4°C, rinsed in phosphate-buffered saline, dehydrated, and embedded in paraffin according to standard techniques. Paraffin-embedded tissues were sectioned (5 µm), exposed to two changes of histoclear, and rehydrated in a series of graded alcohols to water before immunostaining. Lungs from PEG-ADA–treated mice were collected and processed in a similar manner.

RNA Isolation and Cy3- and Cy5-Labeled Amino Allyl–Modified cDNA Probe Synthesis
Total RNA was isolated from Day 5 whole lung tissue using the Trizol reagent from Gibco/BRL (Life Technologies, Grand Island, NY). Total RNA was DNase-treated using DNA-free to eliminate potential genomic DNA contamination (Ambion, Austin, TX). Reverse transcription reactions were performed as described by DeRisi and coworkers with modifications (16). Briefly, total RNA (20 µg) and oligo-dT (2 µg) were incubated at 70°C for 10 min and quick-chilled on ice. Superscript II reverse transcriptase (600 U) and Superscript II reaction buffer (Invitrogen, Carlsbad, CA); dATP, dCTP, and dGTP (0.5 mM each), 0.2 mM dTTP, and 0.3 mM aminoallyl-dUTP (Sigma-Aldrich, St. Louis, MO) were added and the mixture was incubated at 42°C for 2 h. RNA was degraded by the addition of 10 µl 1N NaOH, 10 µl 0.5M EDTA, 25 µl HEPES pH7.0 and heating to 70°C for 15 min. Amino allyl–modified cDNA was purified using GFX purification columns according to manufacturer guidelines (Amersham, Piscataway, NJ). After resolubilization in 0.1 M sodium bicarbonate buffer pH 9.0, cDNAs were coupled to N-hydroxysuccinimidyl esters of Cy3 or Cy5 dyes (CyScribe; Amersham) for 1 h in the dark. The reactions were quenched with 4 M hydroxylamine (Sigma) and unincorporated dyes were removed from CyDye-labeled cDNA using Qia-quick PCR purification columns (Qiagen, Valencia, CA).

Array Hybridization
Spotted long oligonucleotide arrays were produced in the Functional Genomics Core Facility of the UCSF Sandler Center for Basic Research in Asthma1 (San Francisco, CA). Probes consisted of 7,056 oligonucleotides, most of which were 70-mers (Operon Mouse Genome Oligo set V1.1; Qiagen). Arrays were washed five times with distilled, filtered water and dried for 5 min at 600 rpm in a centrifuge. A solution of 0.1% sodium dodecyl sulfate (SDS), 5x saline sodium citrate (SSC), and1% bovine serum albumin was applied to the arrays for 30 min at 42°C to block nonspecific binding. Arrays were washed five times with distilled, filtered water and dried for 5 min at 600 rpm in a centrifuge. Labeled cDNAs were added to hybridization buffer (2.8x SSC, 0.025 M HEPES pH 7.0, 0.2% SDS, 1µg/µl yeast tRNA, and 0.6 µg/µl mouse Cot-1 DNA), denatured for 5 min at 95°C, and applied to the arrays using a coverslip. Each array was placed in a sealed, humidified Dietech hybridization chamber. Hybridization was performed for 20 h in a 63°C water bath, after which the arrays were washed successively with 1x SSC/0.03% SDS, 0.2x SSC, and 0.5x SSC for 3 min each at room temperature and then dried.

Microarray Data Analysis
Arrays were imaged using Axon Instruments (Foster City, CA) 4000B laser scanner and signal intensities were computed using GenePix Pro 4.0 software. The maximum intensity range was adjusted for each array individually to account for differences in overall hybridization signal. To minimize effects of nonspecific hybridization, various gene spot filters were applied using Generic Pro 4.0 software and data was normalized so that the ratio of the median intensities of all features was equal to one. The arrays were split into two groups of 5 arrays each with group 1 comparing differential gene expression between 5 d old ADA-/- and control lungs and group 2 comparing 5 d old ADA-/- lungs with and with out PEG-ADA. 2,675 genes of 7,056 were selected from group 1 and 2,748 of 7,056 genes were selected from group 2 after applying stringent filtering criteria to exclude non specific hybridization signals. The filtered genes signal intensities were transformed to log2 scale and subjected to statistical analysis using Significant Analysis of Microarrays (SAM) software (17) to select significant genes. Stringency parameters were set such that falsely significant genes were ~10% of total selected genes. 180 genes from group 1 and 515 genes from group 2 were selected as significant by SAM based on delta threshold and false discovery rate criteria. The log2 scale intensities of significant genes selected by SAM were subjected to further analysis using Biomedical Research Branch (BRB)-Array tools.2 Gene spots were normalized by median centering each array and genes were screened by excluding an entire gene from all arrays if the p- value was greater than 0.05. Next, average linkage hierarchical clustering for all 10 arrays with centered correlation as the distance metric was performed (18). Dendogram plots and clusters were viewed using cluster viewer in BRB-Array tools.

SYBR-Green Quantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction
Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the Smart Cycler rapid thermal cycler system (Cepheid, Sunnyvale, CA). Reactions were performed in a 25-µl volume with 0.5 µg RNA from Day 5 lungs, 0.3 µM primers for vascular endothelial growth factor D (VEGF-D) (forward 5'-GGTCCATGTTGGAACGATCT-3' and reverse 5'-ATGCTGAGCGTGAGTCCATA-3') and kinase insert domain protein receptor (KDR) (Flk1) (forward 5'-GCGGAGACGCTCTTCATAAT-3' and reverse 5'-CACTTGCTGGCATCATAAGG-3') and reagents included in Quantitech SYBR Green RT-PCR kit (Qiagen). Amplification was as follows: 50°C for 30 min and 94°C for 15 min, followed by 35 cycles of 95°C for 15 s, 58 or 63°C annealing for 30 s, and 72°C extension for 120 s. Annealing temperature varied with specific primers depending on melt temperature. Detection of fluorescent product was performed at the end of the annealing period. To confirm amplification specificity the PCR products from each primer pair were subjected to melting curve analysis. Data was analyzed using Smart Cycler (Cepheid) analysis software. The presence of amplified products were confirmed when the fluorescent signal exceeded an automatic noise-based defined threshold. To generate a standard curve, PCR amplification was performed with template dilutions for each transcript ranging from 100 pg/reaction to 0.001 pg/reaction. A standard curve was created by the Smart Cycler program (Cepheid) according to it's concentration versus the cycle number where it crosses the treshhold (Ct). The final data were normalized to ß-Actin and are presented as the molecules of transcript / molecules of ß-Actin x 100 (% ß-Actin). Results are expressed as the mean ± SEM.

Taqman Quantitative Real-Time Reverse Transcriptase–PCR
Quantitative real-time RT-PCR was performed using the 7,700 Sequence Detector (Applied Biosystems, Foster City, CA) (19). Specific quantitative assays for surfactant proteins (SP-A: (NM_023134) 690(+) -CTTTGGCCTTCACCCTCTTC; 759(-) -CCG GCACAAACTTCTGTCC; 711(+) FAM-TGACTGTTGTTGCTGGCATCAAGTGC SPB: (NM_147779) 567(+) -GCAACAGCTCCCCATTCC; 629(-) -TGAACCCGCTTGATCA GAGT; 608(-) FAM-CTGCAAAGCCAGCAGAAGGGCAG SP-C: (NM_011359) 81(+) -AGGTCCCAGGAGCCAGTTC; 143(-) -ACGATGAGAAGGCGTTTGAG; 103(+) FAM-ATCCCCTGCTGTCCCGTGCAC SP-D: (BC003705) 735(+) -AACTACAGCGTCTAGA GGTTGCC; 810(-) -TCTCCAACACTTCGGCCAT; 762(+) FAM-CCCACTATCAGAA AGCTGCATTGTTCCC) and adenosine receptors (A1, A2, and A3) (20) were developed using Primer Express software (Applied Biosystems) following the recommended guidelines based on sequences from Genbank. Total RNA was isolated from whole lung tissue using the Trizol reagent from Gibco/BRl followed by DNase treatment. This was followed by cDNA synthesis and real time PCR using established protocols (21). The resulting data were analyzed using SDS software (Applied Biosystems) with ROX as the reference dye. The final data were normalized to ß-Actin and are presented as the molecules of transcript/molecules of ß-Actin x 100 (% ß-Actin). Results are expressed as the mean ± SEM.

Terminal Transferase-Mediated dUTP Nick End Labeling Assay
The terminal transferase–mediated dUTP nick end labeling (TUNEL) assay was used according to manufacturer's guidelines (Roche Diagnostics, Mannheim, Germany). Briefly, sections were dewaxed and rehydrated according to standard protocols, incubated with 20 µg/ml of proteinase K (Roche Diagnostics, Mannheim, Germany) in 10 mM Tris · HCl (pH 7.5) for 15 min at 37°C, then rinsed and incubated with the TUNEL reaction mixture for 60 min at 37°C. In negative controls for TUNEL, the transferase enzyme was omitted. All sections were counterstained with Hoechst to stain nuclei. For quantification of positive TUNEL signals, a minimum of 20 high-power fields (x400 magnification) were viewed per animal. The number of TUNEL-positive cells in each high power field was normalized to the total number of cells (positive for Hoechst stain). Results are expressed as the mean normalized percentage of TUNEL-positive cells. The quantitative analyses were performed blinded to the gestational age and genotype of the animals. Results are expressed as the mean ± SEM.

Evidence of apoptosis was confirmed by an additional independent assay evaluating active Caspase-3 by immunohistochemistry (IHC). Endogenous biotin activity was blocked with avidin and biotin (Biotin Blocking Kit; DAKO Corp., Carpinteria, CA), and endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide for 5–10 min. IHC for active caspase 3 and blocking procedures were followed according to manufacturer's guidelines using rabbit IgG Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Sections were incubated for 1 h with a rabbit anti-mouse active caspase-3 antibody (5 µg/ml; R&D systems, Minneapolis, MN) as primary antibody. Bound anti–caspase-3 antibody was visualized with the use of biotinylated goat anti-rabbit IgG antiserum followed by incubation with avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. The slides were developed using 3,3'-diaminobenzidine-tetrachloride (Sigma Fast DAB; Sigma Chemicals, St. Louis, MO) for 7–10 min. In negative controls for anti–caspase-3 staining, the primary antibody was omitted.

Analysis of Type II Cell Apoptosis
To determine the occurrence of type II cell apoptosis, we combined TUNEL with immunohistochemical detection of type II cells with rabbit anti-human pro–surfactant protein C (SP-C) antiserum (Research Diagnostics, Flanders, NJ). For TUNEL, we used the procedures described above.

After TUNEL, the sections were incubated with polyclonal anti-human SP-C antiserum (1:200 for 2 h in the dark). Bound anti–SP-C antibody was visualized with the use of biotinylated goat anti-rabbit IgG antiserum (Vectastain Elite ABC Kit; Vector Laboratories) followed by incubation with avidin-biotinylated peroxidase complex (Vector Laboratories) for 45 min. The slides were developed using Sigma Fast DAB (Sigma Chemicals) for 7–10 min. In negative controls for anti–SP-C staining, the primary antibody was omitted. To further validate the results of the double-immunostaining procedures, sequential sections were independently stained for TUNEL and SP-C with the same chromogens as in the double-staining procedure.

Detection of Cell Proliferation Using In Vivo Bromodeoxyuridine Labeling
Bromodeoxyuridine (BrdU) incorporation into the DNA of proliferating cells was assessed by injecting Day 0, 5, and 10 ADA-/- and control mice, with and without PEG-ADA with 10 µl/gm BrdU labeling reagent (Roche Diagnostics, Mannheim, Germany). Lungs were harvested 2 h later and processed as described above. BrdU labeling was determined using an in situ cell proliferation kit, Fluos, according to manufacturer's instructions (Roche). Briefly, sections were incubated with trypsin solution for 15 min at 37°C and denatured with 4M HCl for 20 min at RT. The sections were then rinsed and incubated with anti-BrdU-FLUOS antibody for 45 min at 37°C. For quantitation of BrdU positive signals, a minimum of 20 high-power fields (x400 magnification) were viewed per animal. The number of BrdU positive cells in each high-power field was normalized to the total number of cells (positive for Hoechst stain). Results are expressed as the mean normalized percentage of BrdU-positive cells. The quantitative analyses were performed blinded to the gestational age and genotype of the animals. Results are expressed as the mean ± SEM.

Quantification of Adenosine and 2'-Deoxyadenosine
Five-day-old ADA-/- and control mice with and without PEG-ADA treatment were anesthetized, the thoracic cavity exposed, and the lungs removed and frozen rapidly in liquid nitrogen. Adenine nucleosides were extracted from frozen lungs using 0.4 N perchloric acid and adenosine and 2'-deoxyadenosine were separated and quantified using reversed phase high-performance liquid chromatography (11). Nucleoside levels were normalized to protein content and values are given as nanomoles per milligram of protein (mean ± SEM).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microarray Analysis Identifies Patterns of Gene Expression Associated with ADA Deficiency
In an attempt to identify developmentally regulated genes that were associated with elevations in lung adenosine levels, microarray transcription profiles were obtained for 5-d-old ADA-/- and control mice, with and without PEG-ADA treatment. Significant genes selected by SAM were exported to BRB array tools for hierarchical clustering using centered correlation and average linkage. Cluster analysis was performed on the log transformed values of the ratio of median intensities of significant genes (see MATERIALS AND METHODS). Thus, a highly conservative method was used that calculated the minimum significant fold change differences in gene expression between different experimental groups. Figure 1A represents the 80 genes that were selected by the above methods for cluster analyses. Dendogram for clustering genes split the genes into two clear clusters with fairly tight average correlations (~ 0.8). Cluster # 1 (n = 40) consists of predominantly overexpressed genes in lungs of ADA-/- mice, whereas cluster # 4 (n = 32) consists of predominantly underexpressed genes in ADA-/- lungs (Figure 1B). The image plot of cluster medians illustrates this clearly. Interestingly, gene clusters over or under expressed in ADA-/- mice were restored with PEG-ADA treatment suggesting that differential gene expression in ADA-/- mice is directly regulated by modifying lung nucleoside levels. Figure 2 represents genes regulated >= 1.4 fold (P <= 0.05 by BRB array tools) in Postnatal Day 5 ADA-/- lungs relative to lungs from control or PEG-ADA–treated ADA-/- mice. These genes fell into one of several categories with respect to predicted biologic function. The bar graph in Figure 2A represents those genes decreased > 1.4-fold in ADA-/- mice. The bar graph in Figure 2B represents those genes increased > 1.4-fold in ADA-/- mice. These changes in gene expression are highly reproducible and represent changes in the expression of a variety of molecular markers, including transcription factors, cell surface antigens, cell cycle regulators, cell adhesion receptors, apoptosis regulators, etc. These microarray findings demonstrate that gene expression patterns in ADA-/- lungs, which have characteristically elevated adenosine levels, differ from expression patterns in control lungs. Furthermore, these changes in gene expression can be regulated by PEG-ADA treatment, which has been used effectively to lower adenosine levels. These findings provide insights into differential gene expression involved in regulation of adenosine-mediated defects in alveogenesis.




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  		Figure 1. (A) Clustergrams of stimulated and repressed genes in ADA-/- mice before and after PEG-ADA treatment. Significant genes selected by SAM were exported to BRB array tools for hierarchical clustering using centered correlation and average linkage. Cluster analysis was performed on the log transformed values of the ratio of median intensities of significant genes. Each row represents one gene and each column represents comparative expression of that gene between two samples hybridized on the same chip. Arrays from Postnatal Day 5 mice are presented in the columns and divided into two groups of ADA-/- mice compared with controls (first five columns) and ADA-/- mice treated with PEG-ADA compared with untreated ADA-/- mice (next five columns). Genes that were present at higher levels in the examined group are shown in progressively brighter shades of red, and genes that were expressed at lower levels are shown in progressively brighter shades of green. Genes shown in black were not different from the two groups being compared. Gene names and GenBank accession numbers are listed adjacent to each gene. (B) Median image and profile line plots of clustered genes. Two clear gene clusters with fairly tight average correlations (0.8) are evident from the median image plots. A row in this plot represents a cluster rather than a gene. Cluster #1 (n = 40) consists of mostly overexpressed genes in lungs of ADA-/- mice, whereas cluster #4 (n = 32) consists of mostly underexpressed genes in ADA-/- lungs. The profile line plots for each cluster represents the median log-ratio of that cluster in each array. Gene clusters over- or underexpressed in ADA-/- mice are restored with PEG-ADA treatment.

 



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  		Figure 2. Genes regulated >= 1.4-fold in Postnatal Day 5 ADA-/- lungs relative to control or PEG-ADA–treated ADA-/- lungs. Yellow bars represent gene expression in ADA-/- lungs compared with controls, whereas blue bars represent expression of the same gene in ADA-/- mice treated with PEG-ADA compared with ADA-/- mice. The bar graph in A represents those genes decreased > 1.4-fold in ADA-/- mice. The bar graph in B represents those genes increased > 1.4-fold in ADA-/- mice. The colored bars represent the mean fold changes from five independent experiments ± SEM. All genes were annotated using OPERON Oligo Microarray Database (Mouse Genome Oligo Set V1.1) (http://oparray.operon.com/~operon/mouse).

 
Validation of Key Regulatory Genes in the Lungs of ADA-/- Mice
We investigated the reliability of the microarray data by candidate gene validation using quantitative RT-PCR. Due to our interest in the processes that control secondary septation in alveogenesis, candidate genes chosen for validation included lung angiogenic factors, Figf (VEGF-D), and KDR (Flk1) (Figure 3A) and surfactant proteins A–D (Figure 3B). The interaction of VEGF-D with specific tyrosine kinase receptors, Flk1 and Flt4, is required for the formation and maintenance of alveolar structures. Expression of VEGF-D and KDR was decreased by 2- and 1.6–fold, respectively, in ADA-/- lungs as shown by microarray analysis. Following PEG-ADA treatment in ADA-/- mice, we observed an increase in the expression of VEGF-D and KDR by a factor of 2.8- and 1.5–fold, respectively. As shown in Figure 3A, VEGF-D and KDR expression analysis by real-time RT-PCR was similar to that seen in the microarray analysis. VEGF-D and KDR transcript levels were significantly reduced in Day 5 ADA-/- lungs compared with expression in control lungs. PEG-ADA treatment from birth significantly enhanced VEGF-D and KDR transcript expression in ADA-/- lungs.




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  		Figure 3. (A) VEGF-D and KDR transcript expression is significantly decreased in Postnatal Day 5 ADA -/- mice. SYBR-Green quantitative real-time PCR analysis of VEGF-D and KDR transcripts were performed on Day 5 control and ADA-/- mice, with or without PEG-ADA. Relative mRNA transcript levels for VEGF-D (black bars) and KDR (gray bars) were calculated by dividing VEGF-D and KDR levels by ß-actin levels measured in the same RNA preparations. Values are mean ± SEM, n = 3. *,#P <= 0.05, Student's t test. (B) SP-C transcript expression is decreased significantly in Postnatal Day 5 ADA-/- mice. Taqman quantitative PCR analysis of surfactant transcripts was performed on Day 5 control and ADA-/- mice, with or without PEG-ADA. Relative mRNA transcript levels for surfactants A–D were calculated by dividing surfactant levels by ß-actin levels measured in the same RNA preparations. SP-C transcript levels in Day 5 ADA-/- lungs (open bars), control lungs (black bars), PEG-ADA treatment from birth (hatched bars). Values are mean ± SEM, n = 3. *,#P <= 0.05, Student's t test.

 
Among different surfactant proteins, SP-C is a known marker of type II alveolar cells that are important progenitor cells during alveogenesis (22). SP-C transcript levels were significantly reduced in Day 5 ADA-/- lungs compared with expression of SP-C in control lungs. According to microarray analysis, the expression of SP-C decreased in ADA-/- lungs by 1.5-fold and expression increased by 1.8-fold following PEG-ADA treatment. As shown in Figure 3B, SP-C expression analysis by real time RT-PCR was similar to that seen in the microarray analysis. PEG-ADA treatment from birth significantly enhanced SP-C transcript expression in ADA-/- lungs. We also extended our quantitative PCR analysis to include other surfactant proteins (A, B, and D) which were not present on the arrays. No difference was observed in the expression levels of the other surfactant proteins. These analyses confirmed a high degree of concordance between oligonucleotide array results and conventional expression analysis results, providing a high degree of confidence in our overall data set. Furthermore, these findings demonstrate that key regulators of lung development and maturation are altered in the lungs in association with ADA deficiency.

Apoptosis as a Mechanism for Failed Secondary Septation
Apoptosis and cell proliferation play an important role in the regulation of lung development (23). Microarray analysis revealed that genes regulating apoptosis and proliferation, like tissue transglutaminases (Ttg) and p-53, were differentially expressed and regulated with PEG-ADA enzyme therapy. To determine if there were alterations in cell death and proliferation in ADA-/- lungs, we analyzed cell death by TUNEL assay in Postnatal Day 0, 5, and 10 ADA-/- and control mice with and without PEG-ADA. The TUNEL method provided a means of identifying apoptotic cells in histologic sections and thereby permitted the determination of the number of cells undergoing apoptosis. Figures 4A, 4C, and 4E represent sections from control lungs on Days 0, 5, and 10, respectively. Figures 4B, 4D, and 4F represent sections from ADA-/- lungs on Days 0, 5, and 10, respectively. Cell death was enhanced in control lungs on Day 0, followed by subsequent decline on Days 5 and 10. In contrast, persistent cell death was observed in ADA-/- mice at all time points examined. Significant increases in cell death were observed in ADA-/- lungs compared with control lungs on Days 5 and 10 (Figure 4G). Immunohistochemical staining for active caspase-3 revealed an increase in the number of positive stained cells in ADA-/- mice on Day 5 and Day 10, indicating apoptosis as a likely mechanism of cell death (Figures 5B and 5D). Because differences in cell death were first observed on Day 5, we examined cell death by TUNEL in ADA-/- and control mice treated with PEG-ADA (Figure 6). No difference in cell death was observed between control and ADA-/- mice treated with PEG-ADA on Day 5. However, a significant decrease in lung cell death was observed in PEG-ADA treated ADA-/- mice on Day 5 compared with untreated ADA-/- mice. These findings demonstrate that persistent and enhanced apoptosis might be a likely mechanism underlying the developmental defects seen in ADA-/- lungs. In addition, lowering adenosine levels with PEG-ADA can control apoptosis observed in Day 5 ADA-/- lungs.




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  		Figure 4. Developmental ontogeny of lung cell death in ADA-/- lungs. Lung sections of Postnatal Day 0, 5, and 10 mice (n = 3 each time point) were stained by the TUNEL technique to detect apoptotic cells (green fluorescent nuclei) by direct immunofluorescence. Top panels A, C, and E represent sections from control lungs on Days 0, 5, and 10, respectively. Lower panels B, D, and F represent sections from ADA-/- lungs on Days 0, 5, and 10, respectively. Two sections were examined per lung. (G) Mean normalized percentage of TUNEL-positive cells calculated by dividing the number of TUNEL-positive cells by the total number of cells. Control lungs (black bars) and ADA-/- lungs (open bars). Values are mean ± SEM. *,#P <= 0.05, using Student's t test. Bar in A = 100 µm and applies to all photographs.

 


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  		Figure 5. Immunohistochemistry for active Caspase 3. Lung sections were obtained from Postnatal Day 5 and Day 10 control and ADA-/- mice. Specimens were processed for detection of apoptosis using rabbit anti mouse caspase 3 active polyclonal antibody. (A) Day 5 control lungs showing specific staining for caspase 3 (arrows). (B) Day 5 ADA-/- lungs showing increased number of caspase 3–positive cells. (C) Day 10 control lungs. (D) Day 10 ADA-/- lungs showing increased number of caspase 3–positive cells. The number of caspase 3–positive cells per 40x magnification is similar to the number of TUNEL-positive cells at same magnification. Bar in A = 50 µm and applies to all photographs.

 


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  		Figure 6. PEG-ADA treatment from birth prevents apoptosis in ADA-/- lungs on Day 5. ADA-/- and control mice were treated with PEG-ADA from birth. Lung sections of Postnatal Day 5 mice (n = 3) were stained by the TUNEL technique to detect apoptotic cells by direct immunofluorescence. Data is presented as mean normalized percentage of TUNEL-positive cells ± SEM. *#P <= 0.05 using Student's t test.

 
To identify a population of dying cells in Day 5 lungs, we combined the TUNEL assay with IHC for pro-SPC (Figure 7). SP-C is a known marker of type II alveolar cells and microarray/real-time RT-PCR analysis identified differential SP-C expression between control and ADA-/- mice with and without PEG-ADA (Figure 3B). Double labeling studies identified type II alveolar cells as one population of apoptotic cells present in Day 5 ADA-/- lungs (Figures 7D–7F). Whereas SP-C–positive type II cells were identified in the lungs of control and PEG-ADA treated ADA-/- mice, these cells were not identified as a population of apoptotic cells by double labeling studies (Figures 7A–7C and 7G–7I). These studies indicate an increase in apoptotic type II alveolar cells as a likely mechanism of failed alveolar septation in ADA-/- mice. This is further supported by the prevention of type II cell apoptosis by PEG-ADA treatment allowing normal alveolar septation in ADA -/- lungs.



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  		Figure 7. Double labeling for TUNEL and SP-C confirms type II cell death in ADA-/- mice. Sections were obtained from lungs of Postnatal Day 5 control (A–C) and ADA-/- mice without (D–F) and with (G–I) PEG-ADA treatment. Specimens were immunostained using rabbit anti mouse SP-C antibody (A, D, G) and TUNEL technique to detect apoptotic cells (B, E, H). Black arrows in A, D, and G indicate SP-C–positive type II alveolar cells. Less staining for SP-C was observed in ADA-/- mice compared with control or ADA-/- mice treated with PEG-ADA. White arrows in B, E, and H indicate TUNEL-positive apoptotic cells. More TUNEL-positive staining was seen in ADA-/- lungs. C, F, and I represent merger between SP-C and TUNEL staining. Notice in F some of the SP-C–positive type II alveolar cells are also TUNEL-positive (both black and white arrows). In control and ADA -/- mice treated with PEG-ADA, separate cells are SP-C–positive (black arrows) and TUNEL-positive (white arrows). Inset points to single cell at x100 magnification. Bar in A = 50 µm and applies to all photographs.

 
Examining the developmental ontogeny of lung cell proliferation on Days 0, 5, and 10 revealed significant differences between control and ADA-/- mice beginning on Day 5 (Figure 8). No difference in proliferation was observed on Day 0 (Figures 8A and 8B). Peak proliferation in control mice was observed on Day 5, followed by a subsequent decline on Day 10 (Figures 8C and 8E). This pattern of cell proliferation was similar to those observed in other studies (24, 25). Similar changes in proliferation were observed in ADA-/- lungs on Days 5 and 10 (Figures 8D and 8F). However, the peak in proliferation in ADA-/- lungs was significantly lower than that observed in control lungs (Figure 8G). In contrast, no difference in cell proliferation was identified between control and PEG-ADA–treated ADA-/- mice on Day 5 (Figure 9). However, there was a significant increase in cell proliferation in ADA-/- lungs on Day 5 following PEG-ADA treatment in comparison to untreated ADA-/- mice. These findings indicate that decreased cell proliferation in ADA-/- lungs in association with increased type II cell apoptosis on Day 5 might contribute to abnormal secondary septation in ADA-/- mice.




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  		Figure 8. Developmental ontogeny of lung cell proliferation in ADA-/- mice. Lung sections of Postnatal Day 0, 5, and 10 mice (n = 3 each time point) were stained by direct immunofluorescence for the detection of BrdU-labeled DNA in proliferating cells (green fluorescent nuclei) after in vivo labeling. Top panels A, C, and E represent sections from control lungs on Days 0, 5, and 10, respectively. Lower panels B, D, and F represent sections from ADA-/- lungs on Days 0, 5, and 10, respectively. (G) Mean normalized percentage of BrdU-positive cells calculated by dividing the number of BrdU-positive cells by the total number of cells. Proliferation in control mice represented by black bars and in ADA-/- mice by open bars. Values are mean ± SEM. *#P <= 0.05, using Student's t test. Bar in A = 50 µm and applies to all photographs.

 


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  		Figure 9. PEG-ADA treatment from birth restores lung cell proliferation in ADA-/- lungs. ADA-/- and control mice were treated with PEG-ADA from birth. Lung sections of Postnatal Day 5 mice (n = 3) were stained by direct immunofluorescence for detection of BrdU-labeled DNA in proliferating cells. Data is presented as mean normalized percentage of BrdU-positive cells ± SEM. *#P <= 0.05 using Student's t test.

 
Developmental Changes Are Associated with Varying Nucleoside Levels and Adenosine Receptor Levels
To associate lung developmental changes with varying nucleoside levels, we measured adenosine and deoxyadenosine levels in Day 5 ADA-/- and control lungs, with and without PEG-ADA treatment. Adenosine levels were significantly elevated in ADA-/- lungs in comparison to control lungs (Figure 10A). PEG-ADA treatment prevented elevations in lung adenosine levels. Low levels of deoxyadenosine were detected in Day 5 ADA-/- lungs. Following PEG-ADA treatment, deoxyadenosine was undetectable in ADA-/- lungs. These findings suggest that the consequences of elevated lung adenine nucleosides in ADA-/- mice likely account for the developmental defects observed in ADA-/- lungs.




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  		Figure 10. Developmental changes associated with varying nucleoside levels and adenosine receptor levels. (A) Adenosine (black bars) and deoxyadenosine (gray bars) levels were quantified in the lungs of Postnatal Day 5 control and ADA-/- mice with and without PEG-ADA treatment (n = 5 each group). Mean values are given as nanomoles per mg protein ± SEM. *#P <= 0.05, using Student's t test. (B) Taqman quantitative PCR analysis of adenosine receptor transcripts. Adenosine A1, A2A, A2B, and A3 receptor transcript levels in Day 5 lungs were calculated by dividing specific receptor levels by ß-actin levels measured in the same RNA preparations. A2A receptor expression was significantly decreased in ADA-/- lungs (open bars) when compared with controls (black bars) and restored with PEG-ADA treatment (hatched bars). *#P <= 0.05, Student's t test.

 
We next evaluated the presence of adenosine receptor transcripts by real-time RT-PCR. Adenosine receptors are seven transmembrane spanning receptors that couple to effector systems through heterotrimeric G-proteins (26). Vertebrates possess four adenosine receptors, termed A1, A2A, A2B, and A3 adenosine receptors. Each receptor has a unique affinity for adenosine and adenosine receptor analogs, and they have distinct cellular and tissue distributions. Transcripts for all four adenosine receptors were identified in Day 5 ADA-/- lungs with expression of A1 receptor transcript being the highest (Figure 10B). Of the four adenosine receptors, A2A receptor expression was significantly decreased in ADA-/- lungs (Figure 10B). PEG-ADA treatment restored A2A expression in ADA-/- lungs. These findings suggest that regulating adenosine receptor specific interactions in ADA-/- mice might mediate the differential expression of genes regulating postnatal lung development. Furthermore, these results suggest that regulation of apoptosis and cell proliferation by controlling lung nucleoside levels with PEG-ADA can prevent the abnormal secondary septation observed in ADA-/- lungs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ADA-deficient mice provide a unique system to examine the impact of elevated adenosine on lung development, and establish a role for adenosine signaling in abnormal alveogenesis. ADA enzyme therapy, which is directed at lowering adenosine levels, effectively altered differential gene expression in ADA-/- lungs in association with preventing abnormal alveogenesis (12, 15). To begin dissection of the mechanisms involved, analysis of the consequences of elevated lung adenosine was conducted on Postnatal Days 0, 5, and 10. Emphasis was placed on the mechanisms regulating secondary septation in postnatal alveolar development, which begins on Day 5. Oligonucleotide microarray technology uncovered regulatory pathways involved in adenosine-mediated lung damage, identifying a number of differentially expressed genes that might be regulated by adenosine interactions and hence play a pivotal role in modulating underlying lung pathology. We hypothesized that apoptosis was one of the forces that impacted alveogenesis in ADA-/- mice based on our microarray findings. It was demonstrated that alveolar differences in ADA-/- mice are consequences of enhanced and persistent apoptosis that was evident on Day 5 in association with elevated lung nucleosides. More specifically, type II alveolar cells were found to be a likely target in ADA-/- mice as shown by TUNEL and SP-C assessment. In addition, enhanced apoptosis in ADA-/- lungs was associated with decreases in lung cell proliferation. ADA enzyme therapy efficiently normalized apoptosis and proliferation in ADA-/- mice. In addition, results suggest that elevated nucleosides in Day 5 ADA-/- lungs might disrupt vascular development by inhibiting expression of VEGF-D and KDR receptors that could lead to abnormal vasculogenesis and associated defects in alveogenesis. These adenosine-dependent alterations in lung development were associated with the presence of adenosine receptors in the developing lung. Interestingly, A2A receptor expression was decreased in Day 5 ADA-/- lungs. In summary, our study provides evidence that the consequences of elevated lung adenosine lead to alterations in gene expression, apoptosis, and proliferation in ADA-/- mice that may regulate abnormal alveogenesis.

One of the most impressive features of the microarray results was the clear distinction in differential gene expression patterns in ADA-/- lungs and their subsequent regulation with ADA enzyme therapy. A usual concern with microarray analysis is obtaining data that are both statistically significant and biologically meaningful. Our stringent analysis parameters significantly decreased the total number of relevant and informative genes, but were sufficient to provide meaningful insights into the pattern of developmental gene regulation in ADA-/- lungs. Close inspection of informative genes shed light on important aspects of postnatal alveolar development in ADA-/- mice. Some of these informative genes included key regulatory growth factors and their receptors that have been clearly implicated to play a role in normal lung development (reviewed in Ref. 27). Of interest was the differential regulation of insulin-like growth factors (IGFs), transforming growth factors (TGFs), fibroblast growth factors (FGFs), and vascular growth factors and their binding proteins (BP). Latent TGFBPs are known to be structural components of the extracellular matrix microfibrils, which associate with elastic fibers. Studies targeted to these binding proteins reveal abnormal lung phenotypes with alveolar disruption (28) due to their role as integral components of elastin-containing microfibrils. Elastin deposition has been implicated in other models of abnormal alveogenesis (29). Although our microarray findings revealed differential expression of TGFBPs, measurement of elastin transcripts did not reveal any differences (S. K. Banerjee, unpublished observations). However, most of these growth factors regulate cell proliferation, differentiation, and migration, allowing one to speculate that disruption of these key regulatory processes might play an important role in postnatal alveogenesis in ADA-/- lungs. This is further strengthened by the observation that ADA enzyme therapy, which is directed at lowering nucleoside levels, was effective in regulating the expression of these developmental genes and permitting normal alveogenesis. In support of this was the coordinated regulation of genes regulating cell proliferation and death. Both apoptosis and cell proliferation are important processes regulating postnatal alveolar development, and increased apoptosis with associated decreased proliferation can contribute to pulmonary hypoplasia (30). The expression of Ttg and p-53 by microarray analysis and caspase-3 by IHC were significantly upregulated in Day 5 ADA-/- lungs and normalized with ADA enzyme therapy, clearly implicating cell death and survival as important mechanisms in the observed pulmonary phenotype. In addition, Ttg are known to suppress cell proliferation and facilitate apoptosis in a caspase 3–dependent manner, further validating our microarray findings (31, 32). The results also indicated differential regulation of SP-C expression, suggesting that SP-C producing cells might be a likely target of elevated lung nucleosides in the lungs of ADA-/- mice.

The coordination of cell proliferation, differentiation, and apoptosis is a central theme in organogenesis (33) and postnatal lung development (23). Septation (alveolarization) largely takes place in the terminal third of the airway tree beginning around Postnatal Day 5 (34). Secondary septa, during alveogenesis, are formed by lifting off tissue ridges from the existing inter airway walls (primary septa) (35). Before septation begins, the lung expands for a short period of time, and the cells of the inter airway walls (primary septa) show a peak of proliferation at Day 4 (36). Hence, cell proliferation is likely a prerequisite for new septal formation (36). This is associated with a rapid increase in the percentage of cells undergoing apoptosis in the first day of life, followed by subsequent decline on Days 5 and 10 (37). Although apoptosis and cell proliferation are both important phenomena in normal postnatal lung development, the deregulation of these processes can impact normal alveolar development. The increased and persistent apoptosis observed in ADA-/- lungs likely involves a number of different cell types. Double immunolabeling for TUNEL and SP-C, permitted colocalization of apoptotic cells and type II alveolar cells in ADA-/- lungs. Elevated lung adenine nucleosides in ADA-/- mice might inhibit terminal differentiation or survival of mature type II cells that maintain high levels of SP-C expression. Although it is not clear whether decreased SP-C expression in ADA-/- lungs is due to loss of type II cells or decreased expression in type II cells, our findings support a role for the type II cell in abnormal alveogenesis seen in the lungs of ADA-/- mice. Although it is evident that preventing type II cell apoptosis with ADA enzyme therapy can prevent alveolar defects in ADA-/- lungs, we cannot exclude the role of endothelial cells (EC). The decreased expression of VEGF-D and KDR (Flk1), well-known markers of endothelial cells, in ADA-/- lungs strongly suggest diminished endothelial cell function. VEGF-D is of particular importance due to its high level of expression in the developing lung (38). VEGF-D is a well known mitogen for endothelial cells and via signaling through various VEGF receptors, VEGFR-2 (KDR or Flk1) and VEGFR-3 (Flt4), regulates endothelial cell function (39). VEGF and KDR are decreased in lung hypoplasia (40), and withdrawal of VEGF or inhibition of its receptors leads to endothelial cell apoptosis and subsequent alveolar destruction (6, 41), suggesting that extensive vascular growth accompanies the increase in alveolarization in normal developing lungs.

Adenosine is known to exert differential effects on apoptosis and cell survival depending on specific cell type and receptor activation (9, 42). Extracellular ATP and adenosine are known to induce endothelial cell apoptosis and inhibit endothelial cell proliferation (43). Based on our findings, we can speculate that alterations of adenosine levels in vivo interfere with VEGF mediated EC survival and hence impact vascular integrity and alveolarization. Adenosine is an important regulator of VEGF expression; however, its effects are determined by cell type and specific adenosine receptor engagement (44, 45). Adenosine receptors are expressed on endothelial cells (46), and VEGF is known to regulate endothelial cell survival through the phosphatidylinositol 3'-kinase pathway, which is also accessed by adenosine signaling (47). Here we have shown that adenosine receptors are expressed in the developing lungs of normal and ADA-/- mice, suggesting a role for adenosine signaling in the observed phenotype. The differential regulation of A2A receptor could have various implications. Activation of the A2A receptor plays an important role in cell survival (48) and hence decreased expression in ADA-/- mice can be related to the persistent apoptosis observed. Engagement of these signaling pathways may regulate the expression of VEGF-D and KDR in the lung, which exerts significant effects on the formation of air–blood barrier and pulmonary vasculature development in ADA-/- mice. This theory seems to hold true, as regulating lung adenosine levels with PEG-ADA allowed for normal alveolar development (12), probably involving both alveolar epithelial and endothelial cell function.

Deficiency of lung maturation due to premature birth is a leading cause of morbidity and mortality in the new born period (respiratory distress syndrome, RDS), with sequale that can extend into adulthood (bronchopulmonary dysplasia, BPD) (49). It has been suggested that BPD may be viewed as the consequence of arrested alveogenesis combined with abnormal repair of immature, injured lungs (50). This concept clearly implicates mechanisms of repair to recapitulate fundamental lung development strategies. However, the molecular determinants that modulate the intricate process of secondary septation in postnatal alveogenesis are far from understood. Clarification of the molecular mechanisms that drive secondary septation might contribute to the identification of specific molecular targets for therapeutic intervention to prevent or ameliorate the morbidity from neonatal and chronic lung disease. Moreover, the lung phenotype of the ADA-/- mice closely mimics the pathomorphologic features of targeted disruption of fibroblast growth factor or platelet-derived growth factor signaling resembling the alveolar hypoplasia found in BPD (4, 5). Adenosine is generated in response to lung injury (51), and several studies have suggested that this signaling pathway might play an important role in chronic lung diseases such as asthma (52) and chronic obstructive pulmonary disease (53). Hence, it would be of considerable interest to measure lung nucleosides in animal models of defective alveogenesis and clinical samples of BPD.

The significance of adenosine accumulation in the lung is unclear; however, the finding that ADA-/- mice exhibit abnormal alveolar defects in association with adenine metabolic disturbances supports the hypothesis that regulation of adenosine levels during the perinatal period is important. The use of ADA enzyme therapy was able to prevent the abnormal alveolar development seen. These benefits of enzyme therapy were directly related to the prevention of adenosine and to a lesser extent deoxyadenosine accumulations in the lung, further suggesting that perturbations in signaling pathways involving these nucleosides are important in lung maturation. Elevated lung adenine nucleosides may inhibit septation for several reasons. Elongation of alveolar septa, by forming ridges, requires epithelial cell division. In addition, new septa must be filled with capillaries and fibroblasts, which also require cell replication. Because elevated adenine nucleosides are associated with decreased cell proliferation in our model, they might prevent septation. Although type II alveolar cell apoptosis is one likely mechanism of failed alveogenesis in ADA-/- mice, we cannot rule out endothelial cell abnormalities due to decreased proliferation or increased apoptosis. Elevated lung adenine nucleosides likely exert significant effects on epithelial–mesenchymal interactions, and additional research into the specific signaling pathways involved will reveal their importance in normal and abnormal lung development.


   Acknowledgments
 
The authors thank ENZON Inc. for their gracious gift of Adagen. M.R.B is supported by grants from the NIH (AI43572 and HL61888) and the Sandler Family Supporting Foundation.


   Footnotes
 
1 Functional Genomics Core Facility, Sandler Center for Basic Research in Asthma, UCSF San Francisco, CA. http://www.som.ucsf.edu/departments/asthmagenomics/index.asp Back

2 Simon, R., and Peng, A. BRB-Array tools Users Guide. National Cancer Institute. Internet address: http://linus.nci.nih.gov/BRB-ArrayTools.html Back

Received in original form March 21, 2003

Received in final form May 27, 2003


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 Introduction
 Materials and Methods
 Results
 Discussion
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