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Molecular and Genomic |
1 Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, USA
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Abstract |
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(Received 12 December 2005;
accepted after revision 21 February 2006;
first published online 23 February 2006)
Corresponding author L. Liu: Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078, USA. Email: liulin{at}okstate.edu
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Introduction |
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5% of the lung alveolar surface area. They synthesize, secrete and recycle lung surfactant. They also transdifferentiate into AEC I to repair the alveolar epithelium after lung injury or during normal fetal lung development (Fehrenbach, 2001). In contrast, AEC I are squamous in shape and cover 95% of the surface area of the alveoli. Traditionally, AEC I are thought to simply function as a barrier for gas exchange. Because of the difficulties in isolating and culturing AEC I, relatively little is known regarding their functions (Williams, 2003). Earlier studies have reported methods to isolate AEC I (Picciano & Rosenbaum, 1978; Weller & Karnovsky, 1986) and recently, several laboratories have improved the isolation protocols and obtained relatively pure AEC I (Dobbs et al. 1998; Borok et al. 2002; Chen et al. 2004a). More studies on the identification of AEC I markers (Qiao et al. 2003; Chen et al. 2004b; Dahlin et al. 2004; Gonzalez et al. 2005) and the physiological functions of AEC I (Johnson et al. 2002; Borok et al. 2002; Ridge et al. 2003) have been performed. AEC I are highly water-permeable and may be responsible for the high water permeability between the alveolar airspace and blood vessels (Dobbs et al. 1998). The epithelial Na+ channel and Na+,K+-ATPase have been identified in AEC I, indicating the participation of these cells in ion and fluid transport in the lung (Borok et al. 2002; Johnson et al. 2002; Ridge et al. 2003). In this study, we performed DNA microarray analysis of AEC I and AEC II. Further characterization revealed that apolipoprotein (Apo E) and transferrin are specifically synthesized by AEC I in the lung. Animal studies suggest that AEC I may function as protective cells to prevent alveolar epithelium from oxidative injury. |
Methods |
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The Pan Rat 10K Oligonucleotide Set (MWG Biotech Inc., High Point, NC, USA) contains aminated 50-mer oligonucleotides representing 6221 known rat genes (4825 Unigene entries), 3594 rat expressed sequence tags (ESTs), and 169 Arabidopsis negative controls. The oligonucleotides were suspended in 3 x SSC at a final concentration of 25 µM and printed on epoxy coated slides by an OmniGrid 100 arrayer (GeneMachine Inc., San Carlos, CA, USA). Each oligonucleotide was spotted in triplicate in three identical 18 mm x 18 mm blocks: A, B and C. The spot–spot distance was 160 µm and the space between blocks was 4 mm. The printed slides were air-dried and stored at room temperature until hybridization. Two slides randomly selected from each print batch were stained with SYBR green II for a quality test and showed uniform spot morphology.
Total RNA was extracted from AEC I and AEC II with TRI reagents (Molecular Research Center, Cincinnati, OH, USA). The RNA samples (3 µg each) were reverse-transcribed into cDNA with Alexa 546 (green)- or Alexa 647 (red)-specific primer of the 3DNA 350 expression kit (Genishere Inc., Hatfield, PA, USA), purified with Microcom YM-30 columns, and mixed with an equal amount of 2x formamide hybridization buffer (50% formamide, 6x SSC, 0.2% SDS, and 10x Denhardt solution). The green–red colour paired cDNA sample was denatured at 80°C for 10 min and added to a DNA microarray slide prewashed with 0.2% SDS. An additional slide was placed face-to-face on this slide as a coverslip. This provided six technical replicated data for each gene from each cell preparation (three replicated spots on one slide and two slides for each hybridization). The slides were incubated at 42°C for 48 h, followed by washing and hybridizing with Alexa 546- and Alexa 647-labelled dendrimers. Hybridized slides were scanned twice (55% PMT and 90% PMT with 90% laser power) with ScanArray Express scanner (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). The dye bias was corrected at experimental and data analysis levels. Experimentally, dye-swap approach was applied in the DNA microarray experiment to balance the difference introduced by fluorescence dyes. In total, there were five preparations of AEC I and AEC II. Three AEC II and two AEC I preparations were labelled with the green dye. Two AEC II and three AEC I preparations were labelled with the red dye. Red-AEC I and green-AEC II or green-AEC II and red-AEC I were paired and hybridized to a slide. In the data analysis step, the bias was compensated by normalization using locally weighted scatterplot smoothing (LOWESS) (see below).
Data analysis
Hybridization images were analysed by the software package GenePix pro 4 (Axon Instruments Inc., Union City, CA, USA). The 90% PMT images were used for the alignment of spots with microarray grids and the 50% PMT one for raw data extraction and subsequent analysis. Log2 ratios of AEC I to AEC II were calculated from the background-subtracted mean fluorescence intensities of the respective spots and normalized by LOWESS. The quality of each spot was evaluated based on spot morphology and quality index (0–4) assigned by the software package RealSpot developed in our laboratory (Chen & Liu, 2005). The quality indices of each gene from replicated hybridizations were averaged. The genes with an average quality index of 1.0 or less were filtered and excluded from further analysis. The differentially expressed genes between AEC I and AEC II were identified by significance analysis of microarray (SAM) using a false discovery rate of 0.01 (P < 0.01) (http://www-stat.stanford.edu/~tibs/SAM/). The identified differential genes were visually validated with spot images using RealSpot. The RealSpot links microarray data with spot images in a spreadsheet table and performs data validation via sorting, searching and editing (Chen & Liu, 2005). The inconsistent genes with spot images were filtered. Then, the genes were functionally grouped by gene ontology annotation (http://www.geneontology.org and http://www.rgd.mcw.edu). Additionally, relative gene expression levels were assessed by signal rank in a 10K gene list. The gene with the highest signal was ranked 1, and lowest ranked 10 000. The rank average of a gene in AEC I or AEC II was considered as the relative expression level in the respective cell type.
AEC I and AEC II isolation
AEC I and AEC II were isolated from 250 g male Sprague-Dawley rats according to our recently developed method (Chen et al. 2004a). The Oklahoma State University Animal Use and Care Committee approved all animal surgeries used in this study. Rats were anaesthetized with intraperitoneal injection of ketamine (40 mg (kg body weight (b.w.))–1) and xylazine (8 mg (kg b.w.)–1). A tracheotomy was performed and rats were ventilated with a rodent ventilator. The thorax was opened along the sternum and the ribs separated. The rats were exsanguinated via abdominal aorta transection. The heart was then transected and a catheter placed in the pulmonary artery. After perfusion, the lungs were removed for the isolation of AEC I and AEC II. The purities of the final AEC I and AEC II preparations were > 90% and > 96% as assessed for each cell preparation by immunocytochemistry using anti-T1
antibodies (E11, a kind gift of Dr Mary Williams, Boston University and Dr Antoinette Wetterwald, University of Berne) and the Papanicolaou staining, respectively. Cross-contaminations of AEC I and AEC II were less than 0.5%. The viabilities of both cell preparations were > 95% as determined by trypan blue dye exclusion.
Quantitative real-time PCR
Data validation of DNA microarray hybridization was performed by quantitative real-time PCR using QuantiTectTM SYBR® Green PCR kit as previously described (Chen et al. 2004b). Primers are listed in Table 1. PCR amplification was performed on ABI PRISM 7700 (Applied Biosystems, Foster City, CA, USA). PCR products were purified with GENECLEAN Turbo kit (Qbiogene, Inc., Irvine, CA, USA) for constructing standard curves (10 to 109 copies). All gene copy numbers were normalized to 18S rRNA.
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This was performed as previously described (Narasaraju et al. 2003). Antigen retrieval was carried out by boiling the sections in a microwave for 5 min in 20 mM citrate buffer (pH 6.0) before permeabilization. Primary and secondary antibodies were goat anti-Apo E (1 : 100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-transferrin (1 : 200, Research Diagnostics, Concord, MA, USA), mouse anti-transferrin receptor (1 : 200, BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-LB-180 (1 : 100, Covance, Richmond, CA, CA), mouse anti-T1 (E11, 1 : 100), and Cy3-conjugated anti-mouse or Alexa 488-conjugated anti-goat antibodies (1 : 500).
Western blot
This was done according to our previous protocols (Narasaraju et al. 2003). Primary antibodies used include goat anti-Apo E (1 : 500), mouse anti-basign (1 : 1000, BD Biosciences), mouse anti-CD9 (1 : 1000, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), mouse anti-Sparc (1 : 1000, Developmental Studies Hybridoma Bank, Iowa), rabbit anti-transferrin (1 : 1000), rabbit anti--b-crystallin (1 : 200, Covance), and anti-ß-actin (1 : 4000, Sigma, St Louis, MO, USA). Secondary antibodies were HRP-conjugated anti-goat IgG (1 : 5000) or HRP-conjugated anti-mouse IgG (1 : 10000).
The secretion of Apo E and transferrin from AEC
Because of limited availability of AEC I, we used AEC I-like cells for examining the secretion of Apo E and transferrin (Paine & Simon, 1996). AEC II were seeded onto 70 mm tissue culture-treated plastic dishes at a density of 5 x 106 cells per dish in minimum essential medium (MEM) with 10% fetal bovine serum and cultured for 7 days. The transdifferentiation of AEC II to AEC I-like cells was nearly complete by day 5. The medium was changed after the first 24 h and every 48 h thereafter. On day 0, 3, 5 and 7, the cells were washed with phosphate-buffered saline (PBS) and lysed using the lysis buffer (10 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA and 1 mM phenylmethylsulphonylfluoride, 10 µg ml–1 aprotonin and 10 µg ml–1 leupeptin). The culture medium was collected on day 3, 5 and 7 and concentrated to 400 µl using an Amicon Ultra filtration system (YM3 membrane with a molecular weight cut off of 3000 Da). Apo E and transferrin proteins in the cells and medium were detected by Western blot.
Hyperoxia exposure of animals
Hyperoxia exposure was done according to the previous protocol (Narasaraju et al. 2003). Pathogen-free male Sprague-Dawley rats (250–275 g) were housed for 1 week before use. Rats were placed in a sealed Plexiglas chamber (90 cm x 45 cm x 45 cm) and exposed to > 95% oxygen for 48, 60 and 72 h. Six rats were used for each exposure time. The flow rate was maintained at 8 l min–1 using a flow meter (VacuMed, Ventura, CA, USA). The oxygen concentration was continuously monitored with an oxygen analyser (Vacumed). During the exposure, animals had free access to food and water. Soda lime was used in the chamber to remove CO2. The control rats were kept in room air. In this set of experiments, two rats died during 72 h exposure time and then were discarded. At the end of exposure, the rat lungs were perfused with 50 mM PBS (pH 7.2) and collected as described above. Lungs were lavaged with 5 ml of normal saline, repeated four times so the final lavage volume was 20 ml. Tissue samples were immediately frozen in liquid nitrogen.
Protection of the lung by Apo E and transferrin from the oxidative injury
Pathogen-free male Sprague-Dawley rats (250 g) were housed for 1 week before use. Transferrin (Tf) (1 mg/rat, Research Diagnostics, Inc.), or human recombinant Apo E2 (50 µg/rat, Pan Vera Corporation, Madison, WI, USA) was intratracheally delivered into rat lungs as described (Jou et al. 2000). There were four groups, each with six animals. (i) Control without hyperoxia, (ii) control vehicle (saline or heat-inactivated Tf) with hyperoxia, (iii) Apo E with hyperoxia, and (iv) Tf with hyperoxia. The rats were anaesthetized with an intraperitoneal injection of ketamine (40 mg (kg b.w.)–1) and xylazine (8 mg (kg b.w.)–1). The epiglottis and trachea of the animals were visualized using a modified intubation wedge. The rats were orally intubated using a sterile 18-guage intravenous catheter. Immediately prior to the administration of the proteins, the rats were forced to exhale by circular compression of the thoracic cavity. The proteins in 75–100 µl sterile PBS were administered using a 1-ml syringe. After 1 h, the treated rats were exposed to > 95% oxygen for 60 h as described above.
Measurement of indices of lung injury
At the end of the hyperoxia exposure, rats were anaesthetized with intraperitoneal injection of ketamine (40 mg kg–1) and xylazine (8 mg kg–1). After thoracotomy, pleural fluid was collected. The rats were exsanguinated by abdominal aorta transection before the lungs were perfused with PBS and lavaged with 7 ml of normal saline three times. Lavage solution (21 ml) was centrifuged to remove the cells and was concentrated to the same volume for Western blot analysis.
For measuring lung vascular permeability and wet-to-dry ratio, the animals were anaesthetized as described above after the hyperoxia exposure. Evans blue dye (EBD, 20 mg (kg b.w.)–1) was injected intravenously through lateral tail vein (Schumacher et al. 2003). EBD was dissolved in sterile PBS at a concentration of 25 mg ml–1. The animals were placed in dorsal recumbency and a midline laparotomy–thoracotomy was performed 30 min after the injection. The rats were ventilated using a ventilator and exsanguinated by severing the abdominal aorta. Later pulmonary artery was canulated and the lungs perfused with PBS. Once cleared of the blood, lungs were removed en bloc from the thoracic cavity. Later, left and right lungs were carefully separated, gently blotted and weighed for determination of wet-to-dry ratios and lung vascular permeability, respectively. Left lungs were dried in an oven at 65°C until three consecutively similar weights were obtained (approximately 3 days) and wet-to-dry ratios calculated. Right lungs were homogenized in formamide (4 ml (g wet tissue)–1) and EBD was extracted for 60 h at room temperature. The homogenates were centrifuged at 4000 g for 30 min and supernatants assayed for EBD. The amount of dye was estimated by measuring the absorbance of the supernatants at 620 nm using EBD as standards. We did not find significant blood contaminations and hence no correction for haeme pigment was done. EBD was expressed as micrograms EBD per gram wet tissue. For all of the experiments involving Apo E and transferrin protection, four animals died and were discarded.
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Results |
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2 (Fig. 1B). These genes are listed in online Supplemental material, Tables S1 and S2. To see the distribution of the genes with relative expression levels, we used the whole 10K data set for ranking analysis (Fig. 1C). The genes with a rank of 1–500 were highly expressed, those of 500–1500 were in the middle, and those of 1500–4000 were weakly expressed; the others were not visually detectable. The differentially expressed genes were distributed in all the gene expression levels. More genes were differentially expressed in AEC I or AEC II when the fold change decreased.
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(4.11 ± 1.22), T1 (3.78 ± 1.08) and aquporin 5 (4.99 ± 1.08) were shown to be differentially expressed in AEC I by our 10K microarray data (ratio of AEC I vs. AEC II, means ±S.E.M.). The cell isolation procedure, in particular positive immunoselection, may affect the expression of some genes. Although we cannot rule out the possibility, the appearence of known AEC I marker genes in the AEC I-specific genes of our microarray data set supports the reliability of our microarray data set. Some of the selected target genes were also verified at protein level for the differential expression of AEC I and AEC II in lung tissue (see below), indicating a genuine differential expression at least for these proteins. All of the current methods for the AEC I isolation procedures, including our own, use positive immunoselection with AEC I marker antibodies to obtain a relatively high purity of AEC I (Dobbs et al. 1998; Borok et al. 2002; Chen et al. 2004a). One potential solution for eliminating the effects of the cell isolation procedure on gene expression is to utilize laser captured microdissection. However, application of this approach to lung AEC I has not been reported yet.
We selected 10 AEC I-specific genes for further characterization based on the fold change (log 2 ratio > 2), coefficient of variation (< 40%), and their cellular location (GO information). Real time PCR confirmed nine genes out of 10 (Fig. 2A). In general, real time PCR showed a higher fold change than DNA microarray. The difference in expression measured by microarray and real time PCR data could be due to the difference between the two methods: solid vs. solution hybridization, the choice of some oligos for the microarray not optimized for microarray hybridization, etc. We could not verify neuronal nicotinic acetylcholine receptor subunit 10 (Chrna10), because of its low expression. Relative mRNA levels of these 10 genes are shown in Fig. 2B. Advanced glycosylation end product-specific receptor (Ager, NM_05333) had the highest expression in AEC I, followed by secreted acidic cysteine-rich glycoprotein (Sparc, NM_01265). Platelet-cell surface glycoprotein (CD9, X76489) and basigin (Bsg, NM_01278) had intermediate expression in AEC I. The other four genes, plasma glutathione peroxidase precursor (Gpxp, NM_022639), wap four-disulphide core domain protein (ps20, AF037272), apolipoprotein E (Apo E, S76779) and transferrin (Tf, NM_017055), had a significant amount of mRNA expression in AEC I and their expression appeared to be more specific to AEC I. Chrna10 (NM_053336) and -b-crystallin (M55534) mRNA levels were very low in AEC I. Ager and Sparc have previously been shown to be cell markers of AEC I (Fehrenbach et al. 1998; Shirasawa et al. 2004; Mustafa et al. 2004).
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Apo E and transferrin are primarily synthesized in the liver, but also in other peripheral tissue including the lung (Driscoll & Getz, 1984; Williams et al. 1985; Yang et al. 1997). Transferrin levels in bronchoalveolar lavage (BAL) fluid are higher than that in the plasma (Mateos et al. 1998), indicating its local synthesis, in addition to coming from the plasma through transudation. However, local sources of both proteins in the lung are unknown. Double-labelling of rat lung tissue indicated that Apo E was colocalized with the known AEC I marker T1, suggesting its AEC I localization (Fig. 3A). The control, which omitted both primary antibodies, did not show signals (Fig. 3A). Other controls that omitted one of the primary antibodies only showed signals in one ‘channel’ (filter), but not in another (data not shown). In contrast, transferrin was found to be present in both AEC I and AEC II in the lung tissue as revealed by immunofluorescence (Fig. 3B). However, the microarray and real time PCR data showed that mRNA expression of transferrin was much higher in AEC I than that in AEC II (Fig. 2B). Furthermore, we could not detect transferrin protein expression in the freshly isolated type II cells by Western blot (Fig. 4). One of the possibilities for this discrepancy is that the AEC II signal detected by immunohistochemistry was due to the binding of extracellular transferrin to the transferrin receptor on the surface of AEC II, because transferrin is present in the BAL fluid of the lung (Mateos et al. 1998). Furthermore, transferrin receptors are expressed in AEC II, but not AEC I, as revealed by double-labelling transferrin receptor with LB-180, a type II cell marker (Fig. 3C). These data suggest that Apo E and transferrin are synthesized in AEC I, which may partially account for local sources of these proteins in the alveolar air space.
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Induction of antioxidant proteins is an adaptive and protective mechanism for cells responding to oxidative stress. We have previously shown that hyperoxia exposure causes oxidative stress, inflammation and acute lung injury (Narasaraju et al. 2003). We reasoned that hyperoxia might up-regulate Apo E and transferrin expression in the lung. Therefore, we exposed rats to > 95% oxygen for different times and determined the levels of these proteins in lung tissues by Western blot. After different time points of hyperoxia treatment (48 h, 60 h and 72 h), Apo E and transferrin protein levels in the lung were increased in an exposure time-dependent manner except that Apo E protein level in the lung at 72 h was slightly decreased in comparison with that at 60 h (Fig. 5A and B). There was a gradual increase of Apo E and transferrin proteins in the BAL fluid (Fig. 5C and D). The increase of these proteins in the BAL could be for various reasons such as AEC injury, flooding of serum proteins due to lung injury (in particular at 72 h), or increased secretion of these proteins by undamaged AEC I or other lung cells. In addition to Apo E and transferrin, other AEC I proteins, including -b-crystallin, CD9, Sparc and basigin, were also increased in both lung tissue and BAL fluid in response to hyperoxia exposure. (Fig. 5A–D).
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, as a biochemical index of lung injury (McElroy et al. 1997). In the control rats, very little of T1 protein was detected in BAL fluid. Hyperoxia exposure caused a significant increase of T1 (Fig. 6B and C). However, the increase of T1 was prevented in the Apo E or transferrin-treated rats. Similarly, hyperoxia increased lung wet-to-dry ratio and lung vascular permeability as determined by changes in the extravasation of Evans blue dye (Fig. 6D and E). Once again, the intratracheal instillation of Apo E and transferrin into the lungs significantly inhibited the increase of both indices of lung injury, even though to a lesser extent in comparison with the type I cell marker in the BAL and the amount of pleural fluid. These data suggest that the instillation of Apo E and transferrin into the lung can prevent hyperoxia-mediated lung injury, but it appears that they protect alveolar epithelium more than pulmonary vascular endothelium.
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Discussion |
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The observation that a single intratracheal instillation of Apo E and transferrin into the lungs before exposure of the animals to hyperoxia provided a significant protection against lung injury raises the question of how long these instilled proteins can remain in the lung. Obviously, the clearance rate of these proteins needs to be determined in order to answer this question. If the clearance rates of these proteins were low, they may act as antioxidants as mentioned above. However, if the clearance rates were high, they may initiate or induce the expression of antioxidants or other defence systems in the lung. Similar studies have been performed in the past (White et al. 1987; Tsan et al. 1990; Tsan et al. 1991; Panos et al. 1995; Yasui et al. 2001). For example, one-time intratracheal instillation of activated protein C inhibits bleomycin-induced mouse lung fibrosis up to 20 days (Yasui et al. 2001). A single intratracheal administration of keratinocyte growth factor, interleukin 1, or tumour necrosis factor reduces hyperoxia-induced mortality and lung injury. Some of this protection is partly due to the induction of antioxidants (White et al. 1987; Tsan et al. 1990; Tsan et al. 1990), but some is not (Panos et al. 1995).
In addition to Apo E and transferrin, several AEC I genes identified in this study may also be involved in lung injury and repair, including Sparc, basigin, -b-crystallin and CD9. These genes were up-regulated in our hyperoxia-mediated lung injury model (Fig. 5). Sparc (also called osteonectin, BM40) is a Ca2+-binding stress-related protein and may function as an extracellular modulator of Ca2+ and other cation-sensitive proteins or proteinases that facilitate cellular proliferation in response to injury (Sage, 1992). Accelerated wound closure, a condition contributing to enhanced contractibility, was observed in Sparc-null dermis, resulting from its decreased collagen content (Bradshaw et al. 2002). Sparc accumulation was used as a marker for stromal repair (Bradshaw et al. 2003). Basigin (also called CD147 or extracellular matrix metalloproteinase inducer) is a multifunctional transmembrane protein involved in inflammation and tumour invasion (Muramatsu & Miyauchi, 2003). Basigin induces tumour cell invasion by stimulating the production of matrix metalloproteinases from surrounding fibroblasts (Kanekura et al. 2002). Similarly, basigin released from AEC I may also trigger the production or release of metalloproteinases in AEC I-surrounding fibroblasts. The released metalloproteinases then degrade extracellular proteins in injured cells and facilitate lung repair. -b-Crystallin (heat shock protein 20 or hsp20) is highly expressed in mammal eye lens. Together with hsp27, it may respond against cell stresses (Horwitz, 2003). CD9, a member of the tetraspanin family, is involved in cellular activities such as cell migration, proliferation, adhesion (Murayama et al. 2004), and the formation and preservation of various different membrane complexes consisting of several functional proteins (Kijimoto-Ochiai et al. 2004). Anti-CD9 monoclonal antibodies inhibited cell proliferation, reduced cell viability and induced morphological changes specific to apoptosis (Murayama et al. 2004).
The findings from current studies may be extended to human medicine. First, some of the AEC I-specific genes identified above, in particular those genes expressed on the plasma membrane of AEC I, may be used as lung injury markers. If verified as being expressed in AEC I of human lung, these genes may be used as a diagnostic tool for acute lung injury. One such marker, HTI56, has been identified (Newman et al. 2000). Second, the understanding of functional roles of these genes in animals would give us an insight for further studies in human lung. For example, transferrin was detected in human BAL fluid (Mateos et al. 1998). The findings that Apo E and transferrin protect lung from hyperoxic injury may help in the development of a therapeutic strategy to treat or prevent lung injury caused by oxidative stress.
This study identified genes expressed specifically in AEC I from the global gene expression of freshly isolated AEC I and AEC II using 10K rat DNA microarray. The data suggest that AEC I play active roles in gas exchange, liquid clearance, lung injury and repair, and lung immune responses. Further studies of two of the AEC I genes, Apo E and transferrin, revealed their functions in the cellular defence against oxidative stress. Therefore, the current studies not only reveal a new function of AEC I cells, cellular defence, but also change the concept that AEC I is only a barrier for gas exchange. AEC I are fully functional cells. Future studies should discover other important functions of AEC I.
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Supplemental material |
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Table S1. Alveolar Type I cell-specific genes
Table S2. Alveolar Type II cell-specific genes
This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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Footnotes |
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