Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Raghava Reddy, Sue Buckley, Melissa Doerken, Lora Barsky, Kenneth Weinberg, Kathryn D. Anderson, David Warburton, and Barbara Driscoll

Department of Surgery and Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, University of Southern California School of Medicine, Los Angeles, California 90027

Submitted 20 May 2003 ; accepted in final form 8 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, {alpha}-, {beta}- and {gamma}-catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.

alveolar epithelial cells; hyperoxic injury; E-cadherin; proliferation


TWO DISTINCT TYPES OF EPITHELIAL CELLS, the alveolar epithelial cell type 1 (AEC1) and the alveolar epithelial cell type 2 (AEC2), line the alveolus, the primary gas exchange surface for the lung. The large, flat AEC1 function by transporting oxygen and carbon dioxide across the alveolar space to the capillary beds that surround the alveoli. These cells are presumed to be terminally differentiated and therefore incapable of cell division, features that make them particularly susceptible to irreparable damage (1). The smaller, cuboidal AEC2 contain prominent cytoplasmic lamellar bodies and perform the differentiated function of surfactant synthesis and secretion. Unlike AEC1, AEC2 retain the ability to proliferate. AEC2 have been observed to differentiate into AEC1 in culture and are assumed to be the progenitor pool for AEC1 in vivo (1, 8). Our previous studies using a rat hyperoxia model have shown that exposure to >90% inspired oxygen causes induction of a cell cycle response in AEC2 that results in both rapid cell division and apoptosis in culture (2, 3). Under these conditions, telomerase activity and expression of the telomerase catalytic subunit, TERT, are also upregulated (6). Taken together, these data support the theory that the epithelial progenitor responsible for repair of damaged distal lung epithelium should be contained within the AEC2 compartment (14, 24).

Despite abundant evidence that AEC2 are capable of generating progeny of both AEC1 and AEC2 types, the exact nature and identity of the cell that performs these functions is unknown. No true stem cell has been isolated for lung tissue, as has been defined for the hematopoietic system. Study of the hematopoietic stem cell has led to a defined set of functions for these rare cells (17). In adult tissues, it is assumed that stem cells remain undifferentiated over the lifespan of the organism and divide infrequently. When repopulation and/or repair are required, stem cells will divide asynchronously, producing one daughter stem cell and one cell capable of proliferating rapidly to form a transiently amplifying population that will then give rise to differentiated progeny (16). In development, an undifferentiated cell expressing multiple markers, including those for alveolar epithelial cells [surfactant protein (SP)-A], Clara cells (CC-10), and neuroendocrine cells (calcitonin gene-related peptide) has been observed in the primitive lung epithelium (27). In the maturing lung, these early progenitors eventually produce defined lineages expressing distinct markers (19). In adults, the AEC2 population appears homogeneous when isolated from quiescent, undamaged lung. However, examination of AEC2 for markers of both proliferation and injury after damage showed that the total population harbors at least four distinct subpopulations: 1) those that do not proliferate and appear undamaged, 2) those that proliferate and appear undamaged, 3) those that do not proliferate and are susceptible to damage, and 4) those that proliferate and are susceptible to damage (2).

We speculated that, although a true alveolar epithelial stem cell might be harbored within the nonproliferating, undamaged population, a transiently amplifying population, capable of repair, would be both proliferative and appear resistant to damage. This latter population would therefore possess the characteristics of an AEC2 progenitor cell population, perhaps capable of generating multiple cell types, but presumably not pluripotent. Unlike an immortal, undifferentiated stem cell, it would possess a finite lifespan and might also exhibit some differentiated function. In addition, the number of cells making up this population could be assumed to be far greater than the number of very rare resident stem cells that might exist in the lung. To find a method for physically isolating the transiently amplifying population, we examined total AEC2 from both control and hyperoxic animals for expression of surface markers, including zonula adherens proteins. We found robust expression of E-cadherin, {alpha}-catenin, {beta}-catenin, and {gamma}-catenin, components of intra- and intercellular structures that function in both cell-cell interactions and surface to nucleus cell signaling. Expression of desmoglein, one of the principal glycoproteins of the cell-cell contact complexes known as desmosomes, and pp120, part of the focal adhesion complex, were also observed. Using immunohistochemistry, we observed heterogeneous expression of E-cadherin in cultured AEC2. Fluorescent-activated cell sorting (FACS) analysis of these same cells confirmed that the AEC2 population isolated from hyperoxic animals could be segregated into at least two subpopulations, expressing either low or high levels of E-cadherin on the cell surface. In agreement with this observation, we were able to physically isolate two distinct AEC2 subpopulations using E-cadherin antibody-coated magnetic beads. Both E-cadherin-positive and -negative populations were analyzed for damage, proliferation, and telomerase activity. Herein, we show that the cells of the E-cadherin-negative subpopulation are seemingly damage resistant and proliferative and exhibit high levels of telomerase activity. From these data, we speculate that this subpopulation might harbor the transiently amplifying progenitor subpopulation of AEC2 responsible for repopulation and repair of damaged alveolar epithelium.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperoxia treatment and AEC2 culture. Adult male Sprague-Dawley rats were exposed to short-term hyperoxia, as described previously (2, 3). Briefly, rats were placed in a 90 cm x 42 cm x 38 cm Plexiglas chamber, and exposed to humidified >90% oxygen for 48 h. Control rats were kept in room air during the treatment period. At the end of the exposure period, the animals were anesthetized by intraperitoneal injection of pentobarbital sodium. After thorough exsanguination by normal saline perfusion via the pulmonary artery, lungs were lavaged to remove macrophages and then subjected to elastase digestion for isolation of AEC2. Differential adherence on rat IgG plates was used to eliminate non-AEC2 cells from the preparation. Cells were plated at 2 x 105 cells/cm2 in DMEM with 10% FBS supplemented with antibiotics for 40 h, harvested by trypsinization for TRAP assay and TdT-dUTP nick-end labeling (TUNEL) preparation or incubated for 20 min in PBS/20 mM EDTA, pH 7.4, at 37°C, and then scraped in PBS and washed for surface marker assays. Examination of attached cells isolated by these methods using both Phosphine R staining for lamellar bodies and immunohistochemistry to detect pro-surfactant protein (SP)-C expression confirmed that >95% of attached cells were AEC2.

Western blotting for zonula adherens proteins. AEC2 isolated from control and hyperoxia-treated animals were cultured for 40 h, washed with PBS, and harvested using PBS/20 mM EDTA, pH 7.4, and scraping as described. Cells were lysed using a 30-min incubation on ice in n-octylglucopyraniside (OPG) buffer, followed by boiling and shearing by five successive passages through a 26-gauge needle. Remaining insoluble material was pelleted by centrifugation at 13,000 g. Total soluble cell protein, which included proteins extracted from cell membranes, was then analyzed for surface marker expression by Western blot analysis using a panel of antibodies to zonula adherens proteins (Pharmingen, San Diego, CA). To analyze differences in protein expression between control and hyperoxia-treated samples, specific bands from four separate blotting experiments were subjected to densitometric scanning using NIH Image version 1.63 software (http://rsb.info.nih.gov/nih-image/Default.html).

Immunohistochemical analysis. AEC2 cultured for 40 h in chamber slides were fixed in acetone-methanol (1:1). For labeling, slides were washed and blocked with Cas-Block (Zymed) and then labeled using a monoclonal antibody to E-cadherin (Pharmingen) at a dilution of 1:5,000. Purified mouse IgG (Sigma), also diluted at 1:5,000, was used as the negative antibody control. Immunostaining was visualized using a fluorescein isothiocyanate (FITC)-labeled secondary antibody at 1:125 (Sigma). BSA (1%) and normal rabbit serum (1%) in PBS was used as a diluent for all antibodies. Results were observed under a Leica DMA microscope at x200 magnification.

FACS analysis for surface markers. AEC2 isolated from hyperoxia-treated rats were cultured for 40 h and then harvested from culture flasks using PBS/20 mM EDTA, pH 7.4, and scraping as described. Vigorous trituration was used to break up clumped cells, which were then filtered through 25 µm nylon mesh to achieve a single cell suspension. Cells were washed in PBS/0.1% BSA to remove EDTA and blocked with Cas-Block. Cells were labeled with antibody to E-cadherin and then fixed in FACSLyse (Becton-Dickinson). After being washed further, FITC-labeled secondary antibody (Sigma) was added. After the final labeling step, cells were further fixed in 1% paraformaldehyde for at least 1 h (and up to 24 h) at 4°C. FACS gates were set on cells labeled with FITC-conjugated secondary antibody alone. Samples were analyzed using a Becton-Dickinson FACScan and CellQuest software. For acquisition of FITC-labeled events, a dot-plot was created for fluorescence detector 1 (FL1) vs. fluorescence detector 2 (FL2) (x vs. y), where FITC fluorescence was measured on the x-axis. For these experiments, 10,000 events were acquired before analysis of each sample for distribution of labeled cells that emitted bright or dim fluorescent signals.

Magnetic bead separation of E-cadherin-positive and -negative AEC2. AEC2 were isolated from hyperoxia-treated rats, cultured for 40 h, and then harvested to achieve a single cell suspension in PBS/0.1% BSA, as described previously. Cells were incubated with magnetic beads (Dynal) coated with antibody to E-cadherin according to the manufacturer's instructions. Subpopulations were separated using the magnetic stand supplied with beads into E-cadherin-positive (attached) and E-cadherin-negative (unattached) populations.

FACS analysis for proliferative profile and TUNEL. Cells placed in suspension as a whole population or separated into subpopulations based on E-cadherin expression using antibody-coated magnetic beads were washed with PBS and then fixed with 1% paraformaldehyde in PBS for 15 min on ice. After being washed, cells were incubated in 70% ethanol at -20°C for at least 24 h before TUNEL analysis was performed according to the Apo-Direct kit manufacturer's instructions (PharMingen). Briefly, fixed cells were washed and then incubated with TdT enzyme and substrate (FITC-dUTP) for 1 h at 37°C. After being washed, cells were counterstained using a propidium iodide/RNase solution. Samples were analyzed using a Becton-Dickinson FACScan and CellQuest software. Parameters for TUNEL were set using positive and negative control cells supplied with the Apo-Direct kit. For acquisition, a standard dual-parameter DNA doublet discrimination gating display template was created with the DNA area signal on the y-axis and the DNA width signal on the x-axis. For these experiments, 10,000 events were acquired, and the nonclumped cells were gated for analysis on a second dual-parameter display with DNA (linear red fluorescence) on the x-axis and FITC-dUTP (log green fluorescence) on the y-axis. These data sets were used to both analyze the cell cycle profile of the entire sample and calculate the percentage of TUNEL-positive cells. Events were analyzed for DNA content and cell cycle phase percentages using ModFit LT 2.0 DNA analysis software (Verity Software House, Topsham, ME).

TRAP assay. Sample preparation and TRAP assays were performed according to the TRAP-EZE protocol (Serologicals, Norcross, GA). Briefly, at least 106 cells for each sample were lysed in 1x CHAPS lysis buffer. The lysate was clarified by centrifugation, and protein content was measured. To assess telomerase activity, samples containing 20 ng protein were incubated with a [{gamma}-32P]dATP end-labeled telomerase-specific primer at 30°C for 30 min for telomere primer extension. The telomerase products were then amplified by 30 rounds of two-step PCR (94°C/30 s, 60°C/30 s). The samples were subjected to 12.5% nondenaturing PAGE in 0.5x 45 mM Tris-borate-1 mM EDTA buffer for 1 h at 500 volts. Gels were dried and exposed to X-ray film to visualize the telomerase products. Each assay included a positive control and a PCR contamination control lane, consisting of all sample elements with the exception of cell lysate. All cell samples were individually controlled for nonspecific PCR products by inclusion of a heat-inactivation control, for which identical aliquots of each sample were incubated at 85°C for 10 min to inactivate telomerase.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of zonula adherens proteins in AEC2. AEC2 isolated from hyperoxia-treated and control rats were lysed to obtain total cell protein, including the majority of membrane-bound species, using detachment by scraping, followed by OPG lysis, boiling, fine needle extrusion, and DNase treatment. Blots of total protein, equilibrated for loading, were analyzed for expression of a variety of adherens proteins. Western blotting of these samples using an antibody to proliferating cell nuclear antigen (PCNA) routinely showed increased expression in cells isolated from hyperoxia-treated animals, indicating an increased level of proliferation in that population. Expression in AEC2 isolated from both control and hyperoxic animals showed robust expression of {alpha}-, {beta}-, and {gamma}-catenin, desmoglein, pp120, and E-cadherin (Fig. 1A). No expression of M-, N-, or P-cadherin was observed. Although antibody-reactive bands for R-cadherin could be visualized, expression in the AEC2 lysates was significantly lower than that observed using positive control cell lysate provided by the antibody manufacturer (data not shown). Over the course of four separate blotting experiments, levels of {alpha}- and {beta}-catenin, as well as E-cadherin, were sometimes moderately higher in cells from hyperoxia-treated animals, although the level of these increases varied from sample to sample. The integrated band intensity for each sample was determined by expressing values obtained by densitometric scanning as a ratio of sample band intensity divided by that of an internal actin control band. Mean values for each data set were then normalized using a factor (protein level of internal actin control of a specific blot divided by the protein level of the first blot performed). Use of these factors nullified differences caused by varying film exposure times for each blot. Calculation of the means and SE for each data set then allowed samples to be analyzed by Student's t-test for differences in band intensity between control and hyperoxia-treated samples. With the exception of PCNA, which was expressed at significantly higher levels in cells from hyperoxia-treated animals (P < 0.05), differences in protein expression for all other samples, including E-cadherin and {beta}-catenin, were not significant (Fig. 1B).



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Fig. 1. Expression of zonula adherens proteins and integrins in alveolar epithelial type 2 (AEC2) cells isolated from control and hyperoxia-treated adult rats. Western blotting analysis of AEC2 cell surface markers revealed the surface marker profile of AEC2. A: in this representative blot, cell lysates from control (C) and hyperoxia-treated (H) animals appeared to express a similar pattern of expression for epithelial cell markers E-cadherin, desmoglein, and pp120, as well as {alpha}-, {beta}-, and {gamma}-catenin. A slight increase in the level of expression of E-cadherin and {beta}-catenin observed in the blot presented here was not reproducible. However, proliferating cell nuclear antigen (PCNA) expression was routinely higher in lysates from hyperoxia-treated animals, indicating an increased level of proliferation in this population. For all blotting experiments, actin was used as a loading control. B: densitometry scan analysis of four separate blotting experiments showed no significant difference in band intensity for proteins analyzed in control lysates (left bar for each sample) or lysates from hyperoxia-treated animals (right bar for each sample). The exception was PCNA (sample 7), analysis of which revealed expression levels in hyperoxia samples at significantly higher levels than those observed in controls.

 

AEC2 subpopulations can be distinguished from heterogeneous expression of E-cadherin. One goal for these studies was to determine if the total hyperoxic AEC2 population, which has heretofore shown heterogeneity of response to damage (2) and expression of hTERT (6), would also contain subpopulations that differed in their expression of surface markers, including adherens proteins. Because this would be impossible to determine using Western blotting, we chose to use immunohistochemistry to determine the distribution of protein expression at the single cell level. E-cadherin, which has been shown to respond uniquely to damage in rat and human lung (9, 10), appeared to be the ideal candidate for future experiments in which physical separation of cells based on surface marker expression would be attempted. In fluorescent antibody staining experiments, the expression of E-cadherin was revealed to be heterogeneous, with some cells exhibiting strong, contiguous expression, some with strong expression on discrete portions of the cytoplasmic membrane only, and some exhibiting very low levels of expression overall (Fig. 2A). Although two other surface proteins were prominently expressed on AEC2 (desmoglein and pp120), these proteins were not considered further for these experiments, since immunohistochemical and FACS analysis showed their expression to be so homogenous as to make characterization of subpopulations difficult (data not shown).



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Fig. 2. Establishment of heterogeneous subpopulations of AEC2 based on surface marker expression. A: E-cadherin expression in AEC2 isolated from hyperoxia-treated rats. Cultured cells were fixed and immunostained for E-cadherin expression and examined under fluorescence microscopy. Negative control, AEC2 immunostained using purified mouse IgG (mIgG) at 1:5,000. E-cadherin, AEC2 immunostained using anti-E-cadherin antibody at 1:5,000. Arrow, strong expression of E-cadherin was observed at certain cell-cell contact points. *Number of cells that exhibit reduced E-cadherin expression at the cell surface. This heterogeneous expression of E-cadherin in AEC2 in culture was reproducibly observed. B: FACS analysis of AEC2 isolated from hyperoxia-treated adult rats showed that the total AEC2 population could be separated into discrete populations based on E-cadherin expression. For dot-plot analysis, gates were set on AEC2 labeled with secondary antibody (AB; FITC-labeled goat anti-mouse) alone. When labeled with primary antibody for E-cadherin (E-CAD), the total AEC2 population was distributed into at least two populations. Cells exhibited either dim fluorescence and thus fell into the same area of the dot plot as the secondary antibody control cells, or bright fluorescence, which caused a shift in the dot plot to the right. Thus dim or bright fluorescence could be correlated with low or high levels of expression of the surface marker. C: quantitative analysis of the results of multiple FACS experiments showed that staining with antibodies to E-cadherin produced two populations, either dim (48.94%) or bright (51.06%) for E-cadherin expression (SE ±7.05, n = 8) experiments.

 

For further analysis of the distribution of expression of E-cadherin on AEC2, cells from hyperoxia-treated rats were isolated and cultured for 40 h, labeled with primary antibody to E-cadherin plus an FITC-conjugated secondary antibody, and then subjected to FACS. FACS gates were set on cells labeled with the secondary FITC-labeled antibody alone and were set to exclude double- or higher-order complexes of cells. All data shown were generated from single cell events. Dot-plot FACS analysis revealed that the total hyperoxic AEC2 population was composed of at least two distinct subpopulations based on E-cadherin expression (Fig. 2B). The observed range of fluorescence from dim to bright was broad for both subpopulations, indicating some heterogeneity even within these two groups of cells (Fig. 2B). These data could be quantitated as the number of events falling within the negative control gate vs. the number shifting to the right, indicating increased antibody binding correlating with an increased level of surface marker expression. As summarized in Fig. 2C, staining with antibodies to E-cadherin showed populations that were predominantly dim (48.94%) or bright (51.06%) for E-cadherin expression (SE ±7.05; n = 8) These data reflect the range of expression of the surface molecule on individual cells, indicating, as was observed using immunohistochemistry, quite variable expression among the total population of AEC2 isolated from hyperoxia-treated animals. Similar values to those described in Fig. 2C were obtained when cells were isolated from control animals that breathed room air, indicating that heterogeneous expression of E-cadherin may be an intrinsic feature of AEC2 in culture (data not shown).

Damaged and survivor populations of AEC2 can be distinguished based on E-cadherin expression. Although cells from both hyperoxia-treated and control animals showed similar distribution of E-cadherin expression, our previous data had shown that these two AEC2 populations were not alike (2, 6). Cells cultured from control animals were quiescent and routinely undamaged, whereas cells from hyperoxia-treated animals were proliferative, exhibited notable levels of DNA damage, and exhibited telomerase activity far above the level normally expressed by AEC2. We wished to determine whether, within the total population, any of these features were the result of unique subpopulations newly formed in the face of hyperoxia treatment. We also wished to determine if these subpopulations would segregate with a particular profile of surface marker expression. We thus began analyzing AEC2 isolated from hyperoxia-treated animals and separated on the basis of E-cadherin expression.

AEC2 isolated from hyperoxia-treated animals and cultured for 40 h were prepared as a single-cell suspension as described. Cells were incubated with magnetic beads (Dynal) coated with antibody to E-cadherin, and the total population was then physically segregated into subpopulations by placing tubes in magnetized holders. Cells not attached to beads were removed for analysis while attached cells were washed several times and then eluted from magnetic beads. The two populations thus obtained were designated E-cadherin negative (unattached to antibody-coated beads and therefore expressing no or very low levels of E-cadherin at the surface of the cell, as recognized by the immobilized antibody) and E-cadherin positive (attached to E-cadherin antibody-coated beads through adequate surface expression of E-cadherin). This nomenclature was verified by subjecting segregated cells to surface molecule labeling using E-cadherin antibody and an FITC-labeled secondary antibody. Subpopulations were then analyzed for E-cadherin expression by FACS. As seen in Fig. 3A, the majority of the E-cadherin-negative subpopulation fell mainly within the negative control gate set on cells labeled by secondary antibody alone. In contrast, the E-cadherin-positive subpopulation showed a distinct shift to the right, indicating the majority of cells were labeled by the anti-E-cadherin antibody. In the example shown, 88.3% of the E-cadherin-positive subpopulation was E-cadherin bright, whereas 93.4% of the E-cadherin-negative subpopulation was E-cadherin dim.



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Fig. 3. Separation of AEC2 from hyperoxia-treated animals into apoptotic and nonapoptotic populations by physical separation based on E-cadherin (ECAD) expression. A: AEC2 isolated from hyperoxia-treated animals were harvested from culture and then incubated with magnetic beads coated with antibody to E-cadherin. After detachment of the bound population, cells from E-cadherin-positive and E-cadherin-negative subpopulations were immunostained using antibody to E-cadherin and then fixed and further stained with an FITC-conjugated secondary antibody. These subpopulations were then subjected to FACS analysis to determine the efficiency of separation. In the example presented, although 88.3% of the E-cadherin-positive population stained positively for E-cadherin, only 6.6% of the E-cadherin-negative population did so. B: AEC2 isolated from hyperoxia-treated animals were harvested from culture and then separated using E-cadherin antibody-coated magnetic beads. Cells from the original population, as well as E-cadherin-positive (attached) and E-cadherin-negative (unattached) populations were fixed and subjected to TUNEL to determine the level of single-strand nicks in each population. Gates for FACS analysis of nick end-labeled cells were set on negative (panel 1) and positive (panel 2) control cells provided with the Apo-Direct kit used for the assay. Those cells falling below the set gate were considered undamaged because of low levels of incorporation of fluorescently labeled dUTP. Analysis of the total AEC2 population revealed distinct subpopulations, both positive and negative for DNA damage (panel 3). Cells in the E-cadherin-negative population appeared to have suffered little or no DNA damage because of hyperoxia treatment (panel 4), whereas the E-cadherin-positive subpopulation was composed of a mixture of apoptotic and nonapoptotic cells (panel 5) C: quantitative analysis of several experiments (n = 3) showed the mean level of DNA damage was 53.02 ± 19.12%. The distribution of damaged and undamaged cells in the E-cadherin-positive population accurately reflected the distribution in the original population, with mean levels totaling 52.88 ± 12.02. Using Student's t-test analysis, these values were not significantly different, indicating that the damaged cells observed in the original population all came from the E-cadherin-positive subpopulation. In addition, the fact that there was no increase in the level of DNA damage indicated that the process of harvesting cells and physically separating them had no further deleterious effects on the cells. The mean number of TUNEL-positive cells in the E-cadherin-negative population was 0.97 ± 0.67%. The difference in DNA damage observed between E-cadherin-positive and -negative subpopulations was highly significant (*P < 0.025).

 

To analyze these subpopulations for functional differences, cells were segregated as described and then fixed, subjected to TUNEL, and assayed for DNA damage by FACS. Analysis was restricted to single cell events, excluding double- and higher-order complexes of cells. As seen in Fig. 3B, gates for TUNEL-negative (panel B.1) and -positive (panel B.2) cells were set on control cells provided with the Apo-Direct kit used for the assay. In this particular example, TUNEL on the original AEC2 population revealed 28% DNA-damaged cells (panel B.3), while, in contrast, the amount of TUNEL-positive cells observed in the E-cadherin-negative subpopulation was 1.3% (panel B.4). The E-cadherin positive subpopulation showed a distribution of damaged and undamaged cells similar to the original, nonsegregated population. For the example in Fig. 4A, that total was 29.33% TUNEL-positive cells (panel B.5).



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Fig. 4. Separation of AEC2 from hyperoxia-treated animals into proliferative and nonproliferative populations by physical separation based on E-cadherin expression. Both the total AEC2 population and subpopulations separated based on E-cadherin expression were fixed and labeled using propidium iodide and then analyzed for DNA content by FACS. Cell cycle analysis was then performed using ModFit software, and the percentage of the total population in G0/G1, S, and G2/M phases was calculated. As an indicator of the proliferative status for each population, the percentage of cells in S phase was compared. The total AEC2 population isolated from hyperoxia-treated rats is proliferative, with a mean S phase percentage at 25.68 ± 3.78% (n = 3). The E-cadherin-negative population exhibited an even more proliferative profile, with the mean number of cells in S phase at 51.59 ± 11.50%. This number was significantly higher than the total obtained from analysis of the total population (*P < 0.05). Conversely, the E-cadherin-positive population was significantly less proliferative than the total population, with the number of cells in S phase averaging 12.8 ± 3.66% (**P < 0.025).

 

Figure 3C presents a statistical analysis of TUNEL-positive cells averaged from three separate experiments (2 animals pooled/experiment, n = 3). The mean number of TUNEL-positive cells for the total AEC2 populations examined in all three experiments was 53.02 ± 19.12%. The distribution of TUNEL-positive and -negative cells within the AEC2 subpopulations based on E-cadherin expression was quite reproducible, with the mean number of TUNEL-positive cells in the E-cadherin-negative subpopulation totaling 0.97 ± 0.67%, whereas the mean number of TUNEL-positive cells in the E-cadherin-positive subpopulation totaled 52.88 ± 12.02%. A comparison of the total AEC2 population and the E-cadherin-positive subpopulation showed little difference in the distribution of damaged and undamaged cells before and after magnetic bead separation, indicating that this process had no substantial DNA-damaging effect on the AEC2. In fact, the difference in the level of damage between the original populations and the E-cadherin-positive populations was not statistically significant, according to analysis using the Student's t-test. Conversely, the difference in DNA damage observed between the original, unsorted population and the E-cadherin-negative subpopulation was highly significant (P < 0.025).

Thus separation of AEC2 from hyperoxia-treated animals on the basis of E-cadherin expression revealed a discrete subpopulation of damaged cells that routinely fell within the E-cadherin-positive subpopulation. Conversely, those cells that appeared undamaged by hyperoxia and culture were invariably contained within the population of cells expressing low levels of E-cadherin.

Proliferative and nonproliferative AEC2 populations can be distinguished based on E-cadherin expression. After isolation and culture as previously described, AEC2 segregated into E-cadherin-negative and -positive populations were fixed and then labeled with propidium iodide. An assay for DNA content was performed using FACS. Data were analyzed using ModFit LT 2.0 DNA software (Verity Software House), which gave values for percentages of cells in G0/G1, S, and G2 + M phases of the cell cycle for each sample. To determine the proliferative status of each population, we compared the numbers of cells in S phase for each group (Fig. 4). Consistent with our previously published data (2, 3), the total AEC2 population isolated from animals exposed to hyperoxia exhibited a proliferative profile, with a mean percentage of cells in S phase of 25.68 ± 3.78% for three experiments in which cells from two different animals were pooled (n = 3). Although this increase in proliferation is routinely observed after hyperoxia treatment, the source of the increase, whether resulting from changes in a discrete subpopulation or an alteration in the behavior of the total hyperoxic population, has not been completely resolved. Immunostaining of cultured AEC2 using antibodies to proliferation markers PCNA and the catalytic subunit of telomerase, hTERT, has indicated the existence of distinct subpopulations expressing higher levels of these markers (6). Intriguingly, upon antibody-bound magnetic bead separation based on E-cadherin expression, the most proliferative cells appeared to segregate in the E-cadherin-negative subpopulation. These cells exhibited a proliferation profile significantly higher than that of the original population, with the mean number of cells in S phase totaling 51.59 ± 11.5% (P < 0.05). Conversely, the E-cadherin-positive population harbored cells that were significantly less proliferative, indicating that the more quiescent cells within the total population segregated into this group. The mean percentage of cells in S phase in the E-cadherin-positive population totaled 12.8 ± 3.67% (P < 0.025). These data indicate that a distinct and highly proliferative subpopulation arises within the total AEC2 population after hyperoxia treatment and is distinguished by its low level of E-cadherin expression.

AEC2 populations expressing high and low levels of telomerase activity can be distinguished based on E-cadherin expression. Telomerase, the enzyme complex responsible for maintaining the integrity of chromosomal ends after division, has been shown to be vital for the continued function of proliferating cells. Our previous data showed that telomerase function and expression of its catalytic subunit, TERT, are upregulated in AEC2 isolated from animals exposed to hyperoxia (6). We postulated that this response might signal the activation of a transiently proliferating population, in which induction of telomerase activity would be required for the proper functioning of cells responsible for damage repair. Once isolated from hyperoxia-treated animals as described, the original population, as well as the E-cadherin-negative and -positive subpopulations of AEC2, was analyzed for telomerase activity. Cells lysates were used as a source of telomerase in the TRAP assay. Samples were run using dilutions of each lysate, from 2 to 200 ng to calibrate the sensitivity of the assay. For the experiments described, 20-ng samples from three separate experiments were analyzed, with each sample representing cells pooled from two individual animals (n = 3). In the example in Fig. 5A, consistent with our previous observations, ample telomerase activity was observed in the total AEC2 population isolated from hyperoxia-treated animals. A similar level of activity was observed in the E-cadherin-negative population, indicating that the majority of cells responsible for the telomerase activity observed in the total sample was harbored within this subpopulation. Conversely, those cells positive for E-cadherin expressed very little telomerase activity, as evidenced by the much lower number of oligonucleotides added to the telomere template. The values for telomerase function obtained in these experiments were analyzed, and the results are presented in Fig. 5B. With the use of the formula TR = n + 3, where TR is the number of telomeric repeats and n is the number of bands that can be counted for each sample, correlating to the number of primers added to the template, we observed that the mean TR value for the E-cadherin-negative population (15.2 ± 0.8) did not differ significantly from the value obtained from the original, unsegregated AEC2 population (17.3 ± 1.4). However, the activity in the E-cadherin-positive population was significantly lower, where the mean TR was 8.1, with an SE of ±3.2 (P < 0.025). Unlike the result obtained from analysis of the proliferative status of the E-cadherin subpopulations, the level of telomerase activity in the E-cadherin-negative subpopulaton never exceeded that of the original, unsegregated population.



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Fig. 5. Separation of AEC2 from hyperoxia-treated animals into populations exhibiting high and low levels of telomerase activity by physical separation based on E-cadherin expression. A: samples from the total AEC2 population and E-cadherin-positive and -negative subpopulations were analyzed for telomerase activity using the TRAP assay. In accordance with the highly proliferative nature of AEC2 isolated in animals exposed to hyperoxia, the telomerase activity in this total population is reproducibly high (lane 1). In contrast, the level of telomerase activity in the E-cadherin-positive subpopulation was quite low (lane 2). Telomerase activity in the E-cadherin-negative subpopulation was at much higher levels (lane 3). B: to quantitate TRAP assay results obtained from several experiments (n = 3), telomerase activity was given a numeric value using the formula telomeric repeats (TR) = number of bands + 3. For the total AEC2 population from hyperoxia-treated rats, the mean TR was 17.3 ± 1.4. This number was similar to that obtained analyzing the E-cadherin-negative population, 15.2 ± 0.8. However, the activity in the E-cadherin-positive population was significantly lower, where the mean TR was 8.1 ± 3.2 (*P < 0.025).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Taken together, the data presented here indicate that AEC2 isolated from rats treated with hyperoxia express distinct surface marker profiles and that the total AEC2 population can be segregated functionally based on the expression of one of those markers, E-cadherin. As summarized in Fig. 1, the zonula adherens expression profiles of cells isolated from hyperoxic and control animals do not appear markedly different. Both populations express the expected cytoskeletal components ({alpha}-, {beta}-, and {gamma}-cadherin) and epithelial cell markers (E-cadherin, desmoglein, and pp120), but not those associated with other cell types (M-, N-, or P-cadherin). However, these expression profiles, analyzed using Western blotting of total AEC2 lysates, could not reveal differences within the total population at the single cell level. Using the single cell analysis approaches of immunohistochemistry and FACS, we were able to observe heterogeneous subpopulations within the total AEC2 population based on a single marker, E-cadherin (Fig. 2).

To determine if the differences between subpopulations were functional, we extended the single cell analysis by using antibody-coated magnetic beads. This method allowed the physical separation of subpopulations. We used separation based on E-cadherin expression in the first instance, both to simplify the experimental approach and since labeling and FACS experiments using this antibody revealed that truly discrete subpopulations could be found within the AEC2 total population based on expression of this marker. At this point, we also chose to focus on cells isolated from hyperoxia-treated animals, since those had previously been shown to contain distinctive subpopulations, whereas the cells from control animals were observed to be composed of a much more homogenous population, typically quiescent and undamaged (2).

Our experiments showed that the E-cadherin-negative population isolated from hyperoxia-treated animals was routinely and on average 99% negative for DNA damage, whereas the E-cadherin-positive population was composed of a mixture of TUNEL-positive and -negative cells, the percentage of each population essentially reflecting that of the original, presegregated population. These data showed a clear difference between the hyperoxic E-cadherin-positive and -negative subpopulations, indicating that discrete subpopulations of AEC2 respond uniquely to oxygen exposure and subsequent subculture on plastic. For example, AEC2 vulnerable to the most long-term DNA-damaging effects of hyperoxia are harbored within the subpopulation expressing high surface levels of E-cadherin. Conversely, those cells that maintain low levels of E-cadherin expression under these conditions are either significantly less likely to undergo damage in culture or maintain an ability to repair damage that is lacking in the E-cadherin-positive population. From analysis of the DNA content of each subpopulation, we concluded that the E-cadherin-negative population harbored the majority of cells capable of proliferation within the total AEC2 population, whereas the E-cadherin-positive population was composed mainly of cells incapable of cycling upon exposure to damage signals. In concordance with this, upon assaying these subpopulations for telomerase activity, we concluded that some portion of the cells expressing low levels of E-cadherin was responsible for most of the telomerase activity observed within the total AEC2 population. This subpopulation, although quiescent in control animals, could therefore be the source of those transiently amplifying cells observed on exposure to damage and thought to perform the function of lung repair, as each challenge would require a new round of replication, dependent on telomere maintenance for proper function.

Results from these studies were obtained using isolated and cultured type 2 cells, and the effects of culture on outcome cannot be disregarded. Although these results reflect observations made in vivo on the effects of hyperoxia, where proliferation and damage are both observed (1, 18), it must be noted that our previous studies have shown that culture on plastic enhances the level of damage observed using TUNEL analysis (2). In addition, we have no information on the role of E-cadherin expression in the lung under hyperoxic conditions, nor whether the expression pattern we observe is a product of cell culture. The procedure used to isolate AEC2 (elastase digestion in situ) effectively strips cells of surface molecule expression, such that analysis of fresh isolates is impossible using antibody-based methods. We speculate that, based on the end-point data we have obtained on damage and proliferation, the E-cadherin expression observed after 40 h in culture could be intrinsic to the individual cell type. However, we recognize that removal of AEC2 from the lung environment and alteration of the matrix and cell-cell signals that ordinarily regulate E-cadherin expression could have a serious impact on its expression profile. As for the role of this molecule in normal lung in vivo, little is known, although it has been reported using immunoelectron microscopy that the molecule is preferentially expressed on AEC2 in normal rat lung, with enhancement of expression on AEC1 observed after injury (9).

Data presented herein do confirm our previous observations that the AEC2 population isolated from hyperoxia-treated rats is strikingly heterogeneous. We have routinely observed that damage and proliferative responses to exposure to oxygen are not a function of the AEC2 population as a whole (2, 6). We now show that subpopulations responsible for these functions can be physically separated from each other based on surface E-cadherin expression. These experiments reveal at least two markedly different subpopulations for AEC2: one comprised of cells that are quiescent, susceptible to damage, and that express very low levels of telomerase activity vs. one comprised of cells that are proliferative, seemingly resistant to damage, and expresses high levels of telomerase activity.

We are currently attempting to establish the parameters of progenitors or progenitor-like cells that may be harbored within the E-cadherin-positive and -negative subpopulations. Although beyond the scope of this study, determination of a regulatory role for E-cadherin in AEC2 progenitor cell function is a next logical step that could lead from our findings. Much of the evidence for a role for E-cadherin in cellular proliferation and survival has been gathered through the study of tumor cells and cell lines, yet recent studies in other systems have found a role for this cellular adhesion molecule in normal cell growth and repair. For example, alterations in E-cadherin expression can be detected after nerve repair and during healing in a skin wounding model, leading to the speculation that regulation of E-cadherin may be required for tissue regeneration and repair (12, 22). Manipulation of E-cadherin activity can have a profound effect on alveolar formation in the mammary gland (5).

Because we observed little difference in the distribution of expression of E-cadherin between control and normal cells, we speculate that changes in the activity of E-cadherin in these two samples may depend on the function of other molecules that interact with E-cadherin. These molecules may respond to hyperoxia treatment by altering their own function as well as that of E-cadherin. Although used in our current studies mainly as a tool for subpopulation selection, the surface protein E-cadherin has a well-established role in epithelial cell proliferation, survival, and migration. A member of the cadherin family, E-cadherin functions in both cell-cell interactions and the intracellular signaling that controls both proliferation and migration. E-cadherin is a surface molecule that interacts with the actin filaments of the cytoskeleton via {beta}-catenin and {gamma}-catenin, which directly bind E-cadherin, and {alpha}-catenin, which in turn links {beta}-catenin and {gamma}-catenin to the actin filaments that make up the cellular cytoskeleton. Spatial or temporal alterations in expression of any of these components can have a marked impact on cellular survival, proliferation, and migration (11). In the case of E-cadherin itself, loss or downregulation of expression in multiple epithelial cell tumor models, including lung cancers, correlates with loss of cellular adhesion and gain of migratory function and a metastatic phenotype (21). Ectopic overexpression of E-cadherin cDNA can reduce the invasive quality of a variety of lung cancers, whereas knockdown of E-cadherin expression results in the transformation of noninvasive carcinoma cell lines to an invasive phenotype (13, 15, 26). In adherent cells, E-cadherin and {beta}-catenin often act in a coordinate manner to regulate cell-cell contact through desmosomes (8). In epithelial cells, attachment of the E-cadherin-{beta}-catenin complex through {alpha}-catenin to actin allows these proteins to regulate cytoskeletal architecture. However, E-cadherin and {beta}-catenin can exist and behave independently of one another in situations of proliferation, migration, and/or stress. Both proteins have been shown to transduce a variety of signals to the nucleus and, as such, are of great interest when examining the fate of cells in response to damage signals, such as those generated by treatment with hyperoxia. {beta}-Catenin itself is intimately involved in the regulation of E-cadherin expression and activity. {beta}-Catenin couples cell surface signals to the Wnt signaling pathway and as such has been implicated in the regulation of both stem cell and cancer cell proliferation (4, 23, 25). E-cadherin and {beta}-catenin expression are often observed to have an inverse relationship, in that upregulation of E-cadherin reduces the expression of {beta}-catenin, resulting in the suppression of cell growth, whereas downregulation of E-cadherin increases the expression of {beta}-catenin, inducing cellular proliferation (20). Although we have yet to examine the functional role of {beta}-catenin in our putative transiently amplifying progenitor population, we have made the preliminary observation that {beta}-catenin, like E-cadherin, is heterogeneously expressed within the total hyperoxic AEC2 population.

The constant exposure to environmental insult leads us to speculate that the lung requires a reservoir of proliferative cells capable of surviving injury and migrating to damaged tissue. By definition, this progenitor population may have retained characteristics required for normal lung development and would play a critical role through the lifespan of the organism for performing lung repair. The established paradigm posits that epithelial injury selectively targets AEC1, whereas epithelial regeneration and repair are performed by AEC2. Although AEC2 as a total population do not conform to the strict definition of a stem cell, experimental evidence suggests that the AEC2 total population could harbor such a cell, as well as the transiently amplifying progeny typically produced by asymmetric stem cell replication in response to injury. We currently lack evidence of AEC2 subpopulations exhibiting pluripotency, immortality, and/or existence in an undifferentiated state. Nevertheless, the AEC2 total population is still considered the best candidate for the regenerative reservoir of the distal lung (14). Our current data show that AEC2 separated on the basis of presence or absence of E-cadherin expression can also be characterized for markers of proliferation and susceptibility to apoptosis. Thus we believe we have made a preliminary step toward isolating an AEC2 subpopulation with a phenotype characteristic of the putative adult lung progenitor.


    ACKNOWLEDGMENTS
 
We thank Prasadarao Nemani for critical reading of this manuscript.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant R01 HL-65352 to B. Driscoll.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Driscoll, Childrens Hospital Los Angeles Research Institute, Smith Research Tower, MS 35, 4650 Sunset Blvd., Los Angeles, CA 90027 (E-mail: bdriscoll{at}chla.usc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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