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Am J Physiol Lung Cell Mol Physiol 291: L1142-L1149, 2006. First published August 4, 2006; doi:10.1152/ajplung.00054.2006
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Galectin-1 in secondary alveolar septae of neonatal mouse lung

Jennifer J. Foster,1 Kelli L. Goss,2 Caroline L. S. George,1 Peter J. Bangsund,2 and Jeanne M. Snyder1,2

Departments of 1Pediatrics and 2Anatomy and Cell Biology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa

Submitted 13 February 2006 ; accepted in final form 18 July 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In mice, alveolarization occurs during postnatal days 4 through 12, when secondary alveolar septae create thin-walled alveoli in the distal lung. We hypothesized that genes predominantly expressed in newly forming secondary alveolar septae influence the process of alveolarization. To address this hypothesis, tips of secondary alveolar septae were isolated from sections of postnatal day 6 mouse lung tissue using laser capture microdissection. Total RNA was isolated and amplified from the dissected alveolar septal tips and from intact postnatal day 6 lung tissue. Gene expression in the samples was characterized using Affymetrix mouse U74AN2 GeneChips. Galectin-1 was an abundantly expressed transcript that was enriched in the alveolar septal tips compared with levels in the whole lung tissue. Galectins are beta-galactoside-binding proteins involved in the regulation of cell proliferation, differentiation, and apoptosis in fibroblasts, muscle cells and endothelial cells, cell types that are present in the alveolar wall. Immunostaining in postnatal day 6 lung tissue confirmed that galectin-1 protein is concentrated in the tips of secondary alveolar septae, predominantly in myofibroblasts. Fibroblasts isolated from day 6 neonatal mouse lung tissue contained galectin-1 protein. Real-time PCR demonstrated that galectin-1 mRNA levels in mouse lung tissue peak at postnatal day 6. Immunoblot analysis confirmed that peak levels of lung galectin-1 protein are found at postnatal days 6 to 12. The increased expression of galectin-1 at the site and time of ongoing alveolarization in the newborn mouse is suggestive that galectin-1 may play an important role in this critical aspect of lung development.

alveolarization; lung development; myofibroblasts; microarray; laser capture microscopy

ALVEOLI CONSTITUTE THE SITE of gas exchange in the lung. The process by which alveoli are formed is not well understood. In humans, the formation of alveoli begins late in gestation and continues through the first few years of postnatal life (24). Prematurely born infants are at risk of developing bronchopulmonary dysplasia (BPD), a disease in which the postnatal formation of alveoli is impaired (16, 17). The number of alveoli is also greatly reduced in emphysema, a lung disease that produces great morbidity and mortality in adults (36). Unfortunately, there are currently no treatments that promote the regeneration of alveoli in damaged lung tissue (2). Thus a better understanding of the process of alveolarization could potentially lead to novel therapies for both BPD and emphysema, as well as for other devastating lung diseases.

In the developing lung, alveoli are created by the growth of secondary septa that divide the terminal air spaces into thin-walled sacs (24). We hypothesized that the tips of secondary alveolar septa contain differentially expressed gene products that mediate alveolarization. We isolated secondary alveolar septal tips from developing mouse lung tissue using laser capture microscopy (LCM), analyzed their gene expression profile, and compared their profile to that of whole lung tissue from day 6 neonatal mice. Using this approach, we determined that several gene products, including galectin-1, are enriched in the tips of alveolar secondary septa.

Galectins are beta-galactoside-binding proteins that are developmentally regulated in several tissues (37). Furthermore, it is known that the galactose-binding lectin activity in rodent lung peaks during the time of alveolarization (7, 39). However, there are at least 10 different mammalian galectins, and the roles of the individual galectin gene products in lung development have not yet been elucidated (37). In the present study of newborn mice, we demonstrate that galectin-1 mRNA and protein are enriched in myofibroblasts present in the distal tips of secondary alveolar septa during the process of alveolarization. In addition, the levels of galectin-1 mRNA and protein in postnatal mouse lung tissue peak during the time of maximal alveolar septation. In contrast, galectin-1 levels are low in adult lung tissue.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Time-dated, pregnant C57/Bl6 mice were purchased from Harlan (Indianapolis, IN). Animals were housed in a pathogen-free environment at the University of Iowa Animal Care Unit, fed Harlan Teklab 7001 mouse chow, and provided water ad libitum. Pregnant mice were killed on day 19 of pregnancy, the fetuses were removed, and fetal lung tissue was dissected. Neonatal mice were killed at postnatal days 6, 12, and 28, and the lung tissue was dissected. Lung tissues from adult mice (at least 7 wk of age) were also collected. This study was approved by the University of Iowa Animal Care and Use Committee.

Tissue. For biochemical measurements, the lung tissues were snap-frozen in liquid nitrogen and stored at –80°C until analysis. For LCM, lung tissue from day 6 neonatal animals was gently inflated via the trachea with a 1:1 mixture of Tissue-Tek OCT compound and phosphate-buffered saline (PBS), cut into pieces, and snap-frozen in liquid nitrogen. Lung tissue for immunohistochemical studies was inflation-fixed via the trachea with 10% zinc formalin (LABSCO, Louisville, KY) at 20 cmH2O pressure and then paraffin-embedded (11).

LCM. Frozen, OCT-inflated lung tissues from day 6 neonatal mice were sectioned 7-µm thick and then thaw-mounted onto glass slides. Slides were stained using the HistoGene LCM Frozen Section Staining Kit (Arcturus, Mountain View, CA) and air-dried. Sections used for collecting secondary alveolar septal tips by LCM were prepared within 1 h of use. CapSure Pads (Arcturus) were used to remove debris from the sections before LCM. Secondary alveolar septal tips were collected with a Pixcell II LCM system and CapSure HS LCM caps (Arcturus). LCM was performed at x40 magnification using a 7.5-µm laser beam, laser power of 75 mV, and a laser duration of 1.5 ms. The laser pulse was centered over the distal aspect of secondary alveolar septae in the neonatal lung tissue. Typically, ~500 secondary alveolar septal tips were collected per LCM cap. Tissue from each frozen section was collected using a new cap; ~20 caps were obtained for each experiment.

RNA isolation, amplification, and hybridization to Affymetrix GeneChips. Ten thousand secondary alveolar septal tips were collected from 10 day 6 neonatal mice, which were obtained from 3 litters. Total RNA from the pooled LCM samples was isolated immediately after each microdissection session (generally from 2 to 3 caps) using an RNAqueous-4PCR kit (Ambion, Austin, TX). Total RNA was also isolated from day 6 neonatal whole lung tissue in parallel. Pooled RNA isolated from the LCM-dissected secondary alveolar septal tips and from the whole lung tissue (2 µg) underwent two rounds of amplification, in parallel, using a RiboAmp RNA Amplification Kit (Arcturus). The amplified RNA was then used to generate cDNA and biotin-labeled cRNA. The biotin-labeled cRNA was hybridized to Affymetrix mouse U74Av2 GeneChips (Affymetrix, Santa Clara, CA). The whole lung tissue and LCM-dissected secondary alveolar septal tip samples were processed simultaneously. Data were analyzed using Affymetrix MAS software. The entire experiment was repeated with tissue obtained from an additional 10 mice obtained from an additional 3 litters. In addition, each individual cRNA sample was hybridized to two different GeneChips. The microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/projects/geo).

Immunohistochemistry. Immunostaining of day 6 neonatal and adult mouse lung tissues was performed using 5-µm paraffin sections that were deparaffinized and rehydrated. For antigen retrieval, the sections were boiled in 0.1 M sodium citrate for 10 min and cooled for 20 min. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in methanol for 5 min. The primary antibodies used were goat anti-mouse galectin-1 (R&D Systems, Minneapolis, MN), rabbit anti-endoglin (R&D Systems), rabbit anti-smooth muscle actin (Abcam, Cambridge, MA), and rabbit anti-mouse surfactant protein B (SP-B; Chemicon International, Temecula, CA). Primary antibodies were diluted to 1:1,000 in sterile PBS. Vector Elite ABC anti-goat or anti-rabbit immunostaining kits (Vector Laboratories, Burlingame, CA) were used, following the manufacturer's instructions. Negative staining controls included sections incubated with PBS or with nonimmune IgG instead of the primary antibody. Immunostained sections were dehydrated, mounted with Permount (Fisher Scientific, Pittsburgh, PA), and photographed using an Olympus microscope (Olympus, Melville, NY) equipped with a digital camera (Spot Jr., Sterling Heights, MI).

In further experiments, double immunostaining was used to colocalize galectin and smooth muscle actin in day 6 neonatal lung tissue. Frozen sections (7 µm thick) were prepared from 50% OCT-inflated day 6 neonatal lung tissue. After fixation in freshly prepared 2% paraformaldehyde for 10 min, sections were rinsed and then incubated in 1% donkey serum in PBS for 1 h at room temperature to block nonspecific antibody binding. The sections were incubated in a mixture of the primary antibodies (both 1:200; goat anti-galectin 1 and rabbit anti-smooth muscle actin) diluted in 1% donkey serum for 1 h at room temperature. After being rinsed, the samples were incubated in a mixture of secondary antibodies (1:200, donkey anti-goat IgG CY5, and donkey anti-rabbit IgG Texas Red) for 30 min at room temperature. After being rinsed, the sections were mounted using Fluormount b and viewed using a Bio-Rad Radiance MRC-1024/confocal microscope (Bio-Rad, Cambridge, MA) operated at the appropriate wavelengths.

Fibroblast culture. Whole lung tissues obtained from postnatal day 6 mice were minced under sterile conditions. The lung tissue was incubated in a 0.25% trypsin-0.01 M EDTA solution (Invitrogen, Carlsbad, CA) for 45 min with trituration every 10 min. The dispersed cells were then centrifuged at 600 g. The resuspended cells were plated in plastic tissue culture dishes in culture medium and allowed to adhere for 2 h. Nonadherent cells were removed and the adherent fibroblasts rinsed with fresh culture medium. This method results in preparations of >95% fibroblasts (6). The cells were cultured in RPMI medium with 10% fetal bovine serum and 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.0025 µg/ml amphotericin B (Invitrogen). Media were changed every 3 or 4 days. The cells were passed after confluence. Experiments were performed using cells from passages 1 or 2.

Immunoblot analysis. Whole lung tissues were obtained from fetal day 19, postnatal days 6, 12, and 28, and adult mice. The tissue was homogenized in PBS that contained 1 mM phenylmethylsulfonylfluoride, a protease inhibitor, and then a 600 g supernatant was obtained. Protein content was assayed using Bradford analysis (5). One hundred micrograms of total protein from each sample was electrophoresed in 15% Tris-glycine gels and then transferred electrophoretically to nitrocellulose membranes. After blocking nonspecific sites on the membrane for 2 h at room temperature in Tris buffer, pH 7.2, containing 7% dry milk and 1% Triton X-100, the membranes were incubated in a 1:1,000 dilution of the goat anti-mouse galectin-1 antibody in Tris buffer with 1% Triton X-100 overnight at 4°C. After three rinses in buffer, the membrane was incubated for 45 min at room temperature in rabbit anti-goat IgG conjugated to horseradish peroxidase (1:8,000; Sigma, St. Louis, MO). The positive signal was detected using an enhanced chemiluminescence kit (ECL; Amersham Biosciences, Buckinghamshire, England). To document equivalent protein loading, membranes were stained with amido black and photographed (26).

Real-time PCR. Total RNA was isolated from whole mouse lung tissues obtained at fetal day 19, postnatal days 6, 12, and 28, and from adult animals using the RNAqueous-4PCR kit (Ambion). Two micrograms of RNA was reverse transcribed at 37°C for 1 h in a total reaction volume of 30 µl that contained 40 units of RNasin (Promega, Madison, WI), 4 µg oligo(dT), 80 units of Moloney murine leukemia virus (MMLV)-reverse transcriptase (Invitrogen), 2.5 mM dNTPs, and 100 mM DTT in MMLV-reverse transcriptase buffer. Real-time PCR was performed using Assay on Demand kits for mouse galectin-1 (Mm 00839408_g1) and GAPDH (Mm 99999915_g1) (Applied Biosystems, Foster City, CA) and a Mx3000P instrument (Stratagene, Cedar Creek, TX). After an initial denaturation step of 95°C for 10 min, 40 cycles of PCR were carried out at 95°C for 15 s and at 60°C for 1 min. Each sample was assayed in triplicate. The relative amount of galectin-1 mRNA was calculated after correction for GAPDH mRNA levels using the comparative threshold cycle (CT) method described in the ABI PRISM 7700 Sequence Detection System User Bulletin no. 2 (Applied Biosystems). As a control, the CT values obtained from real-time RT-PCRs performed with increasing amounts of total RNA and either the galectin-1 or the GAPDH primers were plotted. The two primer sets produced parallel curves with similar slopes.

Statistical analysis. Quantitative data were analyzed using SigmaStat (Jandel Scientific, San Rafael, CA). Depending on the experimental design, data were analyzed using either regression analysis, Student's unpaired t-test, or one-way analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons. Experiments were repeated at least three times unless noted otherwise.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LCM. Alveolarization occurs during postnatal lung development in the mouse, primarily from postnatal days 4 to 12 (1). We used day 6 neonatal lung tissue as a source for collecting the tips of newly forming secondary alveolar septae. As shown in Fig. 1A, secondary alveolar septal tips were collected in frozen sections of the lung tissue by laser capture using a 7.5-µm laser spot. The photographs shown in Fig. 1 were obtained without a coverslip and represent the actual appearance of the lung tissue during the laser capture process. The secondary alveolar septal tips were isolated by lifting the cap off the section. Figure 1B shows the tissue after the cap has been removed. A photograph of the LCM cap shows the dissected secondary alveolar septal tips present on the cap (Fig. 1C). Approximately 10,000 secondary alveolar septal tips were collected on LCM caps, and total RNA was isolated from the pooled LCM-dissected tissue. Because the amount of total RNA obtained from the LCM-dissected samples was too small to measure accurately, it was amplified before use for microarray studies. Total RNA isolated from intact day 6 neonatal mouse lung tissue was amplified in parallel.


Figure 1
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  		Fig. 1. Laser capture microscopy (LCM) of secondary alveolar septal tips in frozen sections of lung tissue obtained from a 6-day-old mouse. All photographs were taken in the absence of a coverslip to demonstrate the actual appearance of the tissue during the laser capture process. A: the lung tissue section has been covered by the LCM cap. The white arrow points to the laser beam. The black arrows indicate secondary alveolar septal tips that have been hit with the laser. B: the lung tissue section after the LCM cap has been removed. Arrows indicate sites where the alveolar secondary septal tips were removed. C: the secondary septal tips have been isolated on the cap. The same captured septal tips shown in the "before" photograph are marked by the black arrows. The laser is indicated by the white arrow.

 
Microchip gene array experiments. We performed two hybridization experiments using Affymetrix U74Av2 mouse GeneChips. In addition, each cRNA sample was hybridized to two different GeneChips to assess the reproducibility of the data. The results from the replicate GeneChips were almost identical (data not shown). Linear regression analysis was used to compare the results of the two independent experiments with respect to gene expression patterns in the secondary alveolar septal tips and in whole neonatal mouse lung tissue (Fig. 2, A and B). There was excellent reproducibility of the signal intensity of the genes detected in the whole lung samples in the two experiments, with a correlation coefficient of 0.920 (P < 0.001) (Fig. 2A). There tended to be more variation observed for genes with lower signal intensities, especially <1,000. The same comparison was performed for the data obtained from the LCM-dissected alveolar secondary septal tips (Fig. 2B). The correlation coefficient for these data was slightly lower, 0.881, but still highly significant (P < 0.001). Again, the greatest variability was observed for genes having signal intensities of less than 1,000. For this reason, to identify genes involved in alveolarization, we only included genes with a signal intensity of <1,000 in the secondary septal tips in all four microchip gene array determinations. We detected 56 genes in the secondary septal tips, which were present according to the Affymetrix software, were also significantly increased in signal intensity in the tips of the secondary alveolar septa compared with their signal intensity in whole lung tissue, and were increased more than twofold in the alveolar secondary septa tips (Table 1). Fifty-one of these genes had been previously characterized (i.e., were not expressed sequence tags). Table 1 shows the 25 genes with the largest increase in signal intensity in the secondary alveolar septal tips compared with their signal intensity in the day 6 whole lung tissue. Expressed sequence tags and five ribosomal protein genes were eliminated from the list. One of the genes identified in this screen was galectin-1, which had a mean signal intensity of ~12,700 in the secondary alveolar septal tips and a mean increase in signal intensity of approximately sevenfold compared with the signal intensity of galectin-1 in the day 6 whole lung tissue (Table 1).


Figure 2
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  		Fig. 2. Regression analyses of microchip gene array data for the whole lung samples (A) and LCM-dissected secondary alveolar septal tip samples (B). Each data point represents the signal intensity of an individual gene product detected in experiment 1 (x-axis) vs. the signal intensity in experiment 2 (y-axis). There are ~10,000 data points in each graph. The signal intensities for the individual gene products were highly reproducible from experiment 1 to experiment 2 (regression analysis, P < 0.001 in both comparisons). Expt, experiment.

 

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  		Table 1. Gene products enriched in secondary alveolar septae of newborn mouse lung

 
Immunostaining. Figure 3 shows the localization of galectin-1 protein in postnatal day 6 mouse lung tissue. Galectin-1 immunostaining was especially concentrated in the distal aspect of secondary alveolar septa in the tissue (Fig. 3A). Intense galectin-1 staining was also present in small crests present on the walls of the primary septa. These small crests may represent newly forming secondary septa (Fig. 3A).


Figure 3
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  		Fig. 3. Immunostaining for galectin-1 in day 6 neonatal mouse lung tissue. Galectin-1 (brown color) was concentrated at the tips of secondary alveolar septae (arrows; A). Staining was also present in small protrusions from the alveolar walls, indicated by asterisks. The staining control (B), i.e., no primary antibody, was negative.

 
Galectin-1 immunostaining was localized to cells in the center of the secondary septae in the neonatal lung (Fig. 4A). Galectin-1 staining was also present in small, newly forming secondary septa (Fig. 4E). Galectin was present in both the nuclei and cytoplasm of positively stained cells (Fig. 4, A and E). Lung sections were also stained for {alpha}-smooth muscle actin, endoglin, and SP-B (Fig. 4). Smooth muscle actin appeared to be present in the same cell type that contained galectin-1 (Fig. 4, B and F). Based on the location, morphology, and expression of smooth muscle actin in this cell type, we identify these cells as myofibroblasts (20). To rule out endothelial cells or epithelial cells as the galectin-1 expressing cell type, some sections were stained for an endothelial cell marker (endoglin) or for SP-B, which is produced by alveolar type II cells (19, 29). The staining patterns observed for these antigens were very different from that observed for galectin-1 and smooth muscle actin (Fig. 4). Endoglin-positive, attenuated endothelial cells (Fig. 4, C and G) were located close to the periphery of the secondary alveolar septa while SP-B positive cuboidal alveolar type II cells were located at the base of secondary alveolar septa (Fig. 4, D and H).


Figure 4
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  		Fig. 4. Localization of antigens in secondary alveolar septae. Paraffin sections of day 6 neonatal lung tissue were immunostained for galectin-1, {alpha}-smooth muscle actin (a myofibroblast antigen), endoglin, (an endothelial cell marker), or surfactant protein B (SP-B), (a type II epithelial cell marker). Galectin-1 was localized in cells within the center of the secondary alveolar septae (A). Actin appeared to be localized in the same cell type as galectin-1 (B). Endoglin was localized in thin cells located close to the periphery of the secondary alveolar septae (C). SP-B was localized in cuboidal type II cells at the base of the secondary alveolar septae (D). Small protrusions into the air space that may represent immature secondary septa were also stained for galectin-1 (E). These small protrusions were also stained for {alpha}-smooth muscle actin (F). Endothelial cells (endoglin positive) were also present at the periphery in these early secondary septae (G). An alveolar type II cell (SP-B positive) was frequently present at the base of small secondary alveolar septae (H). Black arrows indicate positively stained brown cells in A–H. I: immunofluorescence localization (Texas Red) of {alpha}-smooth muscle actin in secondary alveolar septae in a frozen section of day 6 neonatal mouse lung tissue. J: immunofluorescence localization of galectin-1 (CY5, green pseudocolor) in the same tissue shown in J. K: merged images (I and J) reveal colocalization of smooth muscle actin and galectin-1 at the tip of secondary alveolar septae. The tip of the secondary alveolar septae is indicated by a white arrow in I, J, and K.

 
In a further experiment, galectin-1 and smooth muscle actin were colocalized in the day 6 neonatal lung tissue using double immunostaining and confocal microscopy (Fig. 4). Galectin-1 (Fig. 4I) and smooth muscle actin (Fig. 4J) were concentrated at the tip of newly forming secondary alveolar septae. Merging of the galectin-1 and actin images revealed colocalization of the two antigens (Fig. 4K).

Adult mouse lung tissue had very small amounts of galectin-1 protein when immunostained using the same concentration of the primary antibody (1:1,000) used to stain the neonatal lung tissue. In further experiments using less dilute primary antibody (1:500), some galectin immunostaining was detected in the base of alveolar attachments to blood vessels and the pleura and at the distal aspects of some alveolar septae (Fig. 5).


Figure 5
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  		Fig. 5. Localization of galectin-1 in adult mouse lung tissue. Paraffin sections of adult lung were immunostained for galectin-1. Low levels of galectin-1 were localized at attachment sites of alveoli to blood vessels (A) and the pleura (B). In addition, galectin-1 protein was detected at the tips of alveolar septae projecting into the openings of alveolar ducts (AD). TB, terminal bronchiole; RB, respiratory bronchiole; arrows, alveolar septae and positive staining (C).

 
Galectin-1 in cultured neonatal lung fibroblasts. In further experiments, fibroblasts were prepared from day 6 neonatal mouse lung tissue and maintained through two passages to increase their number. Immunoblot analysis showed that the neonatal lung fibroblasts contain galectin-1 protein in amounts comparable to the galectin-1 content of the day 6 neonatal lung tissue (Fig. 6).


Figure 6
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  		Fig. 6. Galectin-1 protein in neonatal lung fibroblasts. Fibroblasts were isolated from day 6 neonatal mouse lung tissue and grown in culture for 2 passages. Homogenate protein from day 6 neonatal mouse lung tissue and cultured neonatal lung fibroblasts were analyzed for galectin-1 protein using immunoblot analysis. The galectin-1 protein migrated at 14 kDa. Comparable amounts of galectin-1 protein were present in the tissue and isolated fibroblasts. The experiment was repeated 3 times.

 
Galectin-1 mRNA and protein. The relative amount of galectin-1 mRNA present in the lung tissue of mice at different developmental stages (fetal day 19, postnatal days 6, 12, and 28, and adult) was determined using real-time PCR. The greatest concentration of galectin-1 mRNA was present in the day 6 mouse lung tissue and varied significantly as a function of developmental age (P = 0.040, 1-way ANOVA) (Fig. 7). The relative amounts of galectin-1 mRNA were much lower in the lungs of 28-day-old and adult mice (Fig. 7). Immunoblot analysis for galectin-1 protein was performed using total lung protein homogenates obtained from fetal day 19, postnatal days 6, 12, and 28, and adult mice. A representative immunoblot is shown in Fig. 8. Using densitometry, it was determined that the highest levels of immunoreactive galectin-1 protein were present at fetal day 19 and neonatal days 6 and 12 (P = 0.012, 1-way ANOVA) (Fig. 8). There were much lower levels of galectin-1 protein present in the adult mouse lung tissue than in neonatal lung (Fig. 8).


Figure 7
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  		Fig. 7. Relative amounts of galectin-1 mRNA in mouse lung tissue at various stages of development. Total RNA was isolated from mouse lung tissue (n = 3) obtained at fetal day 19, postnatal days 6, 12, and 28, and from adult animals (7 wk). The amount of galectin mRNA varied significantly as a function of developmental age (1-way ANOVA, P = 0.04). The greatest amount of galectin-1 mRNA was observed on postnatal day 6 and remained elevated over adult levels on postnatal day 12. The relative amount of galectin-1 mRNA was calculated as described in METHODS. Data (means ± SE) are expressed relative to galectin-1 mRNA levels in the adult lung tissue, which were made equal to 1.

 

Figure 8
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  		Fig. 8. Immunoblot analysis of galectin-1 protein levels in mouse lung tissues. Protein homogenates were obtained from mouse lung tissue obtained at fetal day 19, postnatal days 6, 12, and 28, and from adult mice. Each lane was loaded with 100 µg of lung protein homogenate. A: a representative galectin-1 immunoblot. The inset shows the membrane stained for total protein using amido black and reveals equivalent loading in all lanes. B: the means ± SE of densitometric data obtained from 4 independent experiments. The data are expressed relative to levels of galectin-1 in adult mouse lung tissue, which were made equal to 1. The highest concentration of immunoreactive galectin-1 protein was present at postnatal day 12 (*P = 0.012, 1-way ANOVA, n = 4 per condition).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that galectin-1 mRNA and protein levels are at peak levels in neonatal mouse lung tissue relative to adult levels and are enriched sevenfold in the tips of secondary alveolar septa compared with the galectin-1 content in neonatal whole lung tissue. Immunostaining showed that the galectin-1 protein is localized to the nuclei and cytoplasm of alveolar myofibroblasts. It has previously been reported that the activity of a dimeric galactose-binding lectin, probably galectin-1 based on its molecular weight, peaks during the time of alveolarization in rats, hamsters, and guinea pigs (38). Our data demonstrate that galectin-1 mRNA and protein levels in mouse lung tissue peak during the period of alveolarization, an observation that adds support to the hypothesis that galectins are important in the alveolarization process. Galectin-1 knockout mice have been described as phenotypically normal; however, lung morphology and function have not been investigated in these mice (28).

LCM is a powerful tool that has been used in several pulmonary studies (3, 4, 10, 34). Several investigators have combined LCM with microarray analysis to study gene expression patterns in specific pulmonary cell types or structures (4, 25, 34). The methods for accomplishing this always include amplification of the RNA isolated from LCM-dissected samples because the amount of RNA that can be recovered from the LCM samples is very small (22). In the present study, we used established methods to isolate and amplify RNA from the tips of alveolar secondary septae isolated from neonatal mouse lung tissue. We then compared the gene expression profile of the pooled dissected alveolar septal tips to that of whole lung tissue. Using accepted criteria (i.e., called "present" by the Affymetrix software, a change of greater than 2-fold, and a signal intensity greater than 1,000), we identified genes for which expression was increased in secondary alveolar septal tips. To confirm our findings, we repeated the entire experiment a second time. Based on regression analysis, we found that the data obtained in the two experiments are highly reproducible.

In addition to galectin-1, we identified other genes that were enriched in the tips of the secondary septa, including at least two that have previously been linked to alveolarization, i.e., tenascin-C and drebrin (Table 1) (40, 41). Tenascin-C is an extracellular matrix glycoprotein that promotes a migratory phenotype in surrounding cells and has been linked to the development of the vascular system (14). Tenascin-C protein is concentrated in early postnatal rat lung tissue at the tips of secondary alveolar septa (41). Although tenascin-C null mice apparently develop normally, the fetal lungs of tenascin-C null mice do not undergo normal airway branching morphogenesis (30, 31). In addition, the distal lung air spaces in neonatal tenascin-C null mice are enlarged, an observation suggestive that alveolarization in these animals may be impaired (30). Interestingly, in our microarray screening for genes enriched in the secondary septae, we found that mRNA for another tenascin, tenascin-X, was enriched almost 40-fold in the tips of the secondary septae (Table 1). Tenascin-X probably plays a role in the formation of collagen fibrils (14). Individuals with Ehlers-Danlos syndrome can have mutations in tenascin-X (35). These patients frequently have lung defects, in particular, large cavities in the lungs and a tendency to develop pneumothorax (8, 9, 13). Thus circumstantial evidence may also link tenascin-X to lung alveolarization.

Another gene that we found is enriched in the tips of the secondary alveolar septa is drebrin 1 (Table 1). Drebrin is an actin-binding protein that helps regulate actin filament organization, in particular in cell protrusions of motile cells (33). It has recently been reported that drebrin is located in cell protrusions of {alpha}-smooth muscle actin-expressing cells in the interstitium of the alveolar wall in neonatal rat lung tissue (40). Drebrin is concentrated at the tips of secondary alveolar septa and is not present in adult lung tissue (40).

The tips of secondary alveolar septa include several cell types, including a central core of fibroblasts, myofibroblasts, capillary endothelial cells, and a surface epithelium. We chose to study the tips of the secondary alveolar septa because some of the events that promote the growth and lengthening of the alveolar septa are likely to occur there (23, 27). We identified galectin-1 as a protein for which mRNA is enriched in the secondary alveolar septa, and we confirmed this observation by finding that galectin-1 protein was also concentrated at the tips of secondary alveolar septa. We determined that the galectin-1 protein was present in myofibroblasts in the core of the secondary septa. In addition, galectin-1 and smooth muscle actin were colocalized in this cell type. We identified this cell type as a myofibroblast based on its localization and its {alpha}-smooth muscle actin content. The expression of {alpha}-smooth muscle actin in lung alveolar cells has been linked to a myofibroblast cell phenotype in many studies (20, 21). Fibroblasts isolated from neonatal day 6 lung tissue also express galectin-1, which is consistent with our identification of the galectin-expressing cell type within the secondary septae. Interestingly, galectin-1 protein was present in both the cytoplasm and nucleus of positively stained cells. Galectin-1 has been detected in the nucleus of several other cell types and it has been postulated that galectin-1 may play a role in regulating the transcription of genes involved in differentiation (37). Our findings suggest that galectin-1 is not expressed in endothelial cells of secondary alveolar septae, since the cellular staining pattern for endoglin, an endothelial cell marker, was markedly different than the pattern for {alpha}-smooth muscle actin and galectin-1 staining.

Some galectin-1 protein is also present in adult mouse lung tissue but at much lower levels than observed in the day 6 and day 12 postnatal mouse lung tissues. The adult lung galectin-1 is primarily present at sites of attachment of alveolar septa to the pleura and blood vessels. Galectin-1 immunostaining was also observed in the tips of some alveolar septa in alveolar ducts. The presence of galectin-1 in these sites is suggestive that these locations may be involved in alveolarization in the adult lung or that galectin-1 may play another role in the adult lung.

The function of galectin-1 in the lung has yet to be defined. Galectin-1 has previously been shown to stimulate the transformation of murine dermal fibroblasts to myocytes (12). We speculate that pulmonary galectin-1 may promote the differentiation of neonatal lung alveolar fibroblasts to a myofibroblastic cellular phenotype and via this mechanism stimulate the elongation of the secondary alveolar septa. Additionally, galectin-1 has been shown to increase cell division in cultured vascular endothelial cells (32). Thus another possible role for galectin-1 is stimulation of the growth of capillaries in the developing alveolar secondary septa, an essential process in the formation of an optimal air-blood interface in the forming alveolar wall. Finally, galectin-1 has been shown to induce apoptosis in several cell types (15). Apoptosis occurs in early postnatal rat lung tissue and is increased immediately following the peak of lung alveolarization (6, 18). The highest concentration of galectin-1 protein in postnatal lung tissue is observed at around the end of alveolarization and, therefore, galectin-1 may be involved in the regulation of apoptosis in the developing alveolar wall. We found no evidence of galectin-1 staining in septal capillary endothelial cells or alveolar epithelial cells. Thus we conclude that galectin-1 in the neonatal lung may play a role in myofibroblast growth and differentiation and in this way influence morphogenic events within the growing alveolar secondary septa.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-62861 (J. M. Snyder) and T32-HL-07638 (J. J. Foster) and a Forest Pharmaceuticals Advancing Newborn Medicine Fellowship (J. J. Foster).


   ACKNOWLEDGMENTS
 
We thank Kevin Knudson at the University of Iowa DNA core facility for assistance with the microarray studies, the University of Iowa Department of Pathology for use of a laser capture microscope, and Tom Moniger for assistance with the confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Snyder, Dept. of Anatomy and Cell Biology, Univ. of Iowa Carver College of Medicine, Iowa City, IA 52242 (e-mail: jeanne-snyder{at}uiowa.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amy RW, Bowes D, Burri PH, Haines J, and Thurlbeck WM. Postnatal growth of the mouse lung. J Anat 124: 131–151, 1977.[ISI][Medline]
  2. Barnes PJ and Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985–996, 2004.[CrossRef][ISI][Medline]
  3. Betsuyaku T, Griffin GL, Watson MA, and Senior RM. Laser capture microdissection and real-time reverse transcriptase/polymerase chain reaction of bronchiolar epithelium after bleomycin. Am J Respir Cell Mol Biol 25: 278–284, 2001.[Abstract/Free Full Text]
  4. Betsuyaku T and Senior RM. Laser capture microdissection and mRNA characterization of mouse airway epithelium: methodological considerations. Micron 35: 229–234, 2004.[CrossRef][ISI][Medline]
  5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  6. Bruce MC, Honaker CE, and Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol 20: 228–236, 1999.[Abstract/Free Full Text]
  7. Clerch LB, Whitney PL, and Massaro D. Rat lung lectin synthesis, degradation and activation. Developmental regulation and modulation by dexamethasone. Biochem J 245: 683–690, 1987.[ISI][Medline]
  8. Cupo LN, Pyeritz RE, Olson JL, McPhee SJ, Hutchins GM, and McKusick VA. Ehlers-Danlos syndrome with abnormal collagen fibrils, sinus of Valsalva aneurysms, myocardial infarction, panacinar emphysema and cerebral heterotopias. Am J Med 71: 1051–1058, 1981.[CrossRef][ISI][Medline]
  9. Dowton SB, Pincott S, and Demmer L. Respiratory complications of Ehlers-Danlos syndrome type IV. Clin Genet 50: 510–514, 1996.[ISI][Medline]
  10. Fuke S, Betsuyaku T, Nasuhara Y, Morikawa T, Katoh H, and Nishimura M. Chemokines in bronchiolar epithelium in the development of chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 31: 405–412, 2004.[Abstract/Free Full Text]
  11. George CL, White ML, O'Neill ME, Thorne PS, Schwartz DA, and Snyder JM. Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin. Am J Physiol Lung Cell Mol Physiol 285: L1337–L1344, 2003.[Abstract/Free Full Text]
  12. Goldring K, Jones GE, Thiagarajah R, and Watt DJ. The effect of galectin-1 on the differentiation of fibroblasts and myoblasts in vitro. J Cell Sci 115: 355–366, 2002.[Abstract/Free Full Text]
  13. Herman TE and McAlister WH. Cavitary pulmonary lesions in type IV Ehlers-Danlos syndrome. Pediatr Radiol 24: 263–265, 1994.[CrossRef][ISI][Medline]
  14. Hsia HC and Schwarzbauer JE. Meet the tenascins: multifunctional and mysterious. J Biol Chem 280: 26641–26644, 2005.[Free Full Text]
  15. Hsu DK and Liu FT. Regulation of cellular homeostasis by galectins. Glycoconj J 19: 507–515, 2004.[CrossRef][ISI][Medline]
  16. Husain AN, Siddiqui NH, and Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 29: 710–717, 1998.[CrossRef][ISI][Medline]
  17. Jobe AH and Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163: 1723–1729, 2001.[Free Full Text]
  18. Kresch MJ, Christian C, Wu F, and Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res 43: 426–431, 1998.[ISI][Medline]
  19. Lebrin F, Deckers M, Bertolino P, and Ten Dijke P. TGF-beta receptor function in the endothelium. Cardiovasc Res 65: 599–608, 2005.[Abstract/Free Full Text]
  20. Leslie KO, Mitchell JJ, Woodcock-Mitchell JL, and Low RB. Alpha smooth muscle actin expression in developing and adult human lung. Differentiation 44: 143–149, 1990.[CrossRef][ISI][Medline]
  21. Low RB. Modulation of myofibroblast and smooth-muscle phenotypes in the lung. Curr Top Pathol 93: 19–26, 1999.[Medline]
  22. Luzzi V, Mahadevappa M, Raja R, Warrington JA, and Watson MA. Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J Mol Diagn 5: 9–14, 2003.[Abstract/Free Full Text]
  23. Mariani TJ and Kaminski N. Gene expression studies in lung development and lung stem cell biology. Curr Top Dev Biol 64: 57–71, 2004.[CrossRef][ISI][Medline]
  24. McGowan S and Snyder J. Development of alveoli. In: The Lung: Development, Aging and the Environment, edited by Harding R, Pinkerton KE, and Plopper CG. London: Elsevier Academic, 2004.
  25. Mohr S, Bottin MC, Lannes B, Neuville A, Bellocq JP, Keith G, and Rihn BH. Microdissection, mRNA amplification and microarray: a study of pleural mesothelial and malignant mesothelioma cells. Biochimie 86: 13–19, 2004.[Medline]
  26. Nakamura K, Tanaka T, Kuwahara A, and Takeo K. Microassay for proteins on nitrocellulose filter using protein dye-staining procedure. Anal Biochem 148: 311–319, 1985.[CrossRef][ISI][Medline]
  27. Pierce RA and Michael Shipley J. Retinoid-enhanced alveolization: identifying relevant downstream targets. Am J Respir Cell Mol Biol 23: 137–141, 2000.[Free Full Text]
  28. Poirier F and Robertson EJ. Normal development of mice carrying a null mutation in the gene encoding the L14 S-type lectin. Development 119: 1229–1236, 1993.[Abstract]
  29. Pryhuber GS. Regulation and function of pulmonary surfactant protein B. Mol Genet Metab 64: 217–228, 1998.[CrossRef][ISI][Medline]
  30. Roth-Kleiner M, Hirsch E, and Schittny JC. Fetal lungs of tenascin-C-deficient mice grow well, but branch poorly in organ culture. Am J Respir Cell Mol Biol 30: 360–366, 2004.[Abstract/Free Full Text]
  31. Saga Y, Yagi T, Ikawa Y, Sakakura T, and Aizawa S. Mice develop normally without tenascin. Genes Dev 6: 1821–1831, 1992.[Abstract/Free Full Text]
  32. Sanford GL and Harris-Hooker S. Stimulation of vascular cell proliferation by beta-galactoside specific lectins. FASEB J 4: 2912–2918, 1990.[Abstract]
  33. Shirao T. The roles of microfilament-associated proteins, drebrins, in brain morphogenesis: a review. J Biochem (Tokyo) 117: 231–236, 1995.[Abstract/Free Full Text]
  34. Taatjes DJ, Palmer CJ, Pantano C, Hoffmann SB, Cummins A, and Mossman BT. Laser-based microscopic approaches: application to cell signaling in environmental lung disease. Biotechniques 31: 880–894, 2001.[ISI][Medline]
  35. Uitto J and Ringpfeil F. Ehlers-Danlos syndrome-molecular genetics beyond the collagens. J Invest Dermatol 122: xii–xiii, 2004.[ISI][Medline]
  36. Walter R, Gottlieb DJ, and O'Connor GT. Environmental and genetic risk factors and gene-environment interactions in the pathogenesis of chronic obstructive lung disease. Environ Health Perspect 108 Suppl 4: 733–742, 2000.
  37. Wang JL, Gray RM, Haudek KC, and Patterson RJ. Nucleocytoplasmic lectins. Biochim Biophys Acta 1673: 75–93, 2004.[Medline]
  38. Whitney P, Maxwell S, Ryan U, and Massaro D. Synthesis and binding of lactose-specific lectin by isolated lung cells. Am J Physiol Cell Physiol 248: C258–C264, 1985.[Abstract/Free Full Text]
  39. Whitney PL, Starcher B, and Brittain C. Soluble beta-galactoside specific lectin is developmentally regulated in lungs of neonatal black mice and beige mice. Exp Lung Res 18: 553–561, 1992.[ISI][Medline]
  40. Yamada M, Kurihara H, Kinoshita K, and Sakai T. Temporal expression of alpha-smooth muscle actin and drebrin in septal interstitial cells during alveolar maturation. J Histochem Cytochem 53: 735–744, 2005.[Abstract/Free Full Text]
  41. Zhao Y and Young SL. Tenascin in rat lung development: in situ localization and cellular sources. Am J Physiol Lung Cell Mol Physiol 269: L482–L491, 1995.[Abstract/Free Full Text]



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