AJP - Lung Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L1352-L1360, 2007. First published February 16, 2007; doi:10.1152/ajplung.00328.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/L1352    most recent
00328.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Myerburg, M. M.
Right arrow Articles by Pilewski, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Myerburg, M. M.
Right arrow Articles by Pilewski, J. M.

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

Mike M. Myerburg, Joseph D. Latoche, Erin E. McKenna, Laura P. Stabile, Jill S. Siegfried, Carol A. Feghali-Bostwick, and Joseph M. Pilewski

Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 25 August 2006 ; accepted in final form 14 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.

airway epithelium; ion transport; epithelial-mesenchymal interactions; ciliogenesis

THE EPITHELIAL SURFACE LINING the conducting airways provides a protective barrier from the external environment and clears inhaled and aspirated pathogens from the lung (19). For efficient mucociliary clearance, the bronchial epithelium maintains several properties. First, the apical surface of human bronchial epithelium (HBE) must contain abundant properly functioning cilia that serve to propel overlying mucus from the distal airways to the proximal airways where the mucus can be expelled (51). Second, as the airways are constantly injured by inhaled pathogens, the lining epithelium must be able to rapidly regenerate so that the protective surface is not compromised. Additionally, HBE must be able to properly transport sodium, through the epithelial sodium channel (ENaC), and chloride, through the cystic fibrosis transmembrane conductance regulator (CFTR) and other chloride channels, to maintain the airway surface liquid (ASL) volume required for mucus clearance (28–30). The signaling mechanisms that serve to achieve these phenotypic properties in the epithelial lining surfaces of the airway are not fully understood.

During lung development and during epithelial repair following injury, interactions with subepithelial mesenchymal cells are fundamental to HBE cell migration, proliferation, and differentiation, which are required cellular processes for epithelial restoration (10). During lung development, bidirectional communication within the "epithelial-mesenchymal tropic unit" is necessary for normal bronchial branching and subsequent epithelial differentiation (11). In the developed lung, these epithelial-mesenchymal interactions are important for epithelial repair following injury (18). Known mediators involved in epithelial-mesenchymal interactions include hepatocyte growth factor (HGF), IGF-1, basic fibroblast growth factor, platelet-derived growth factor, endothelin-1, erbB ligands, and transforming growth factor-beta (TGF-beta), as well as extracellular matrix (ECM) proteins (6, 11, 18, 21, 22, 31, 34, 38, 42, 47, 48, 58, 59). Furthermore, subepithelial fibroblast hyperplasia is a common feature of chronic airway diseases (2, 18, 39) such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF), suggesting that alterations in epithelial-mesenchymal interactions contribute to airway disease pathogenesis. Despite the known importance of the subepithelial mesenchyme in epithelial repair and differentiation, little is known how the two cell types communicate.

The goal of HBE cell culture is to recapitulate native airway biology as much as possible so that investigation can be conducted ex vivo in a simplified environment. Initial attempts to establish well-differentiated HBE cultures were hindered by the use of serum-containing differentiation media, which contains TGF-beta that induces terminal squamous differentiation and growth arrest in HBE cells (9, 13, 15, 24, 27). Subsequently, serum-free alternatives were developed that allow for the differentiation of well-polarized, ciliated HBE cell cultures. The most commonly used serum-free differentiation media are 1) bovine pituitary extract-containing media, which is supplemented with various growth factors (see Table 1; Refs. 7, 8, 12, 23, 30, 41, 56), and 2) media containing the proprietary Ultroser G (USG), which is derived in part from calf brain (5, 41, 57). Regardless of the differentiation media used, the ECM composition underlying HBE cells is also important in normal differentiation (4, 37). When cultured in a conducive media at air-liquid interface, HBE cells develop into a pseudostratified polarized layer with apical cilia and basolateral nuclei, which morphologically resembles native airway epithelium. Furthermore, cultured HBE cells exhibit ion transport properties, specifically sodium and chloride conductances characteristic of ENaC and CFTR, that approximate that of explanted native lung (20).


View this table:
[in this window]
[in a new window]

  		Table 1. Essential supplements in serum-free HBE differentiation media

 
In an effort to recapitulate the native airway architecture in vitro, we developed an HBE cell and lung fibroblast coculture system utilizing primary human cells derived from excess pathological lung tissue. Similar systems have been previously reported as effective methods of culturing bronchial and skin epithelial cells (25, 48, 53). In the model recently reported by Wiszniewski et al. (53) and Vermeer et al. (48), a subepithelial fibroblast feeding layer in conjunction with HBE differentiation media augmented HBE cell proliferation, increased Na+ transport, and allowed for normal ciliogenesis and mucin production. We further explored the effects of subepithelial fibroblasts on epithelial phenotype and wound repair and assessed the importance of HGF for fibroblast modulation of epithelial differentiation. We provide evidence that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) HGF is important for subepithelial fibroblast modulation of HBE cell differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All cell culture medium was obtained from GIBCO (Invitrogen, Carlsbad, CA) except bronchial epithelial growth media (Clonetics, San Diego, CA) and USG (BioSepra, Cedex, France). Neutralizing HGF antibodies were kindly gifted from Galaxy Biotech (Mountain View, CA), specifically, mouse monoclonal antibody L2G7 to human HGF- and isotype-matched control antibody (mouse IgG2a) (17). Recombinant human HGF (rhHGF) was obtained from R&D Systems (Minneapolis, MN). Unless otherwise specified, all other reagents were obtained from Sigma.

HBE and lung fibroblast isolation and culture. HBE cells were cultured from excess pathological tissue following lung transplantation and organ donation under a protocol approved by the University of Pittsburgh Investigational Review Board, as previously described (5). Briefly, bronchi from the second to sixth generation were dissected, thoroughly rinsed, and incubated overnight at 4°C in Eagle's minimum essential media. The bronchi were then digested overnight at 4°C in minimum essential media (MEM) containing 0.1% protease type XIV and 0.01% deoxyribonuclease. The epithelial cells were removed from the underlying stroma by blunt dissection, isolated by centrifugation, and washed in MEM containing 5% fetal bovine serum (FBS). After centrifugation, the cells were resuspended in bronchial epithelial growth medium (BEGM) and plated into type VI human placental collagen-treated tissue culture flasks. On reaching 80–90% confluence, the passage 0 cells were trypsinized (0.1%), resuspended in BEGM, and seeded onto human placental collagen-coated Costar Transwell filters (cat. no. 3470, 0.33 cm2, 0.4-µm pore) at a confluent density of ~2 x 105/cm2. Twenty-four hours following seeding, the filters were placed at air-liquid interface in the conditions described below. Primary lung fibroblasts were cultured from ~2 cm3 pieces of explanted peripheral lung. The tissue was minced, and the fibroblasts were cultured in DMEM supplemented with 10% FBS, as previously described (36). Following subculture, the fibroblasts were used in passages 4–8 for coculture experiments. The explanted lung tissue was isolated from patients with COPD, idiopathic pulmonary fibrosis, primary pulmonary hypertension, and CF (genotype {Delta}F508/{Delta}F508). Qualitative differences in epithelial morphology due to disease state were not observed.

HBE differentiation conditions. HBE-fibroblast cocultures were established by seeding ~5,000 fibroblasts on the undersurface of an inverted Transwell insert containing a confluent layer of undifferentiated HBE on the apical surface. After the fibroblasts had adhered to the filter for 30 min, the Transwell insert was placed into DMEM supplemented with 10% FBS and allowed to differentiate at an air-liquid interface. Alternatively, cells were incubated in fibroblast-conditioned media (FCM) that was established by seeding 5,000 fibroblasts in the bottom of the Transwell in DMEM/10% FBS. For control conditions, HBE cells without subepithelial fibroblasts were allowed to differentiate at air-liquid interface with DMEM/F-12 supplemented with 2% USG as the basolateral media. Epithelial-fibroblast cocultures were also established using HBE cells that had been differentiated in USG-containing media, and experiments were conducted following the indicated number of days in coculture. Schematics of the different culture conditions are provided in the Fig. 1 insets.


Figure 1
View larger version (135K):
[in this window]
[in a new window]

  		Fig. 1. Human bronchial epithelial (HBE) cell morphology following 28 days of coculture with subepithelial fibroblasts. Representative confocal images are shown of HBE cells cultured in 2% Ultroser G (USG; A), 10% FBS conditioned by fibroblasts (B), and 10% FBS with subepithelial fibroblasts (C). XZ reconstructions accompany the images to demonstrate cell polarity. D: the subepithelial fibroblast layer has been reconstructed from the confocal images taken of the coculture in C. Cilia and nuclei are labeled by antiacetylated tubulin (green) and DRAQ5 (blue). Cellular cytoskeletal shape (F-actin) is outlined by rhodamine phalloidin (red). Bar, 5 µm; magnification, x400; FCM, fibroblast-conditioned media.

 
In all culture conditions, the basolateral media was changed twice weekly. The rate of media discoloration was similar between control and coculture conditions. In coculture conditions, the subepithelial fibroblasts became confluent after ~1 wk of coculture and a portion of nonadherent fibroblasts grew on the bottom of the well.

Immunohistochemistry. The Transwell inserts were fixed in 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 5% goat serum prior to incubation with antiacetylated tubulin antibodies (Sigma-Aldrich, St. Louis, MO). Species-appropriate secondary antibody conjugated to Alexa 488 (Jackson Research, West Grove, PA) was used to visualize tubulin. Additionally, DRAQ5 (Alexis, Lausen, Switzerland) and fluorescently labeled phalloidin (Molecular Probes, Carlsbad, CA) were used as nuclear and cytoskeletal markers, respectively. Images were taken using an Olympus FluoView 500 confocal microscope and analyzed using a MetaMorph image analysis workstation (Universal Imaging, West Chester, PA). To visualize the epithelial and fibroblast layers independently, the confocal stack was divided at the level of the filter, and each stack was reconstructed separately. To measure the percentage of ciliated cells in the epithelial cell layer, the number of ciliated cells was determined by manually counting random fields and dividing by the total cell count as measured in MetaMorph using automated nuclear counting. In addition to assuring that the investigator was blinded to the conditions, the percentage of pixels occupied by the Alexa 488 was measured in MetaMorph in the same random fields as a complementary method.

Epithelial injury. Mechanical scrape injury was induced by creating a line with a pipette tip across the surface of a confluent HBE cell layer. Following injury, the apical surface was washed with DMEM to remove denuded epithelial cells, and the area of injury was examined to assure uniformity. The HBE cells were serially fixed over 72 h following injury and visualized using F-actin (Molecular Probes) immunohistochemistry. Additionally, the rate of wound closure was assessed by serial measurements of transepithelial resistance (RTE) using a Millicell electrical resistance system (Millipore, Bedford, MA). Before RTE measurements, the apical surface of the HBE was submerged in DMEM/F-12 to create an electrical circuit.

Short-circuit recordings. Short-circuit currents (Isc) were measured as previously described (3, 5, 32). In brief, cells cultured on filter supports were mounted in modified Ussing chambers, and the cultures were continuously short-circuited with an automatic voltage clamp (Department of Bioengineering, University of Iowa, Iowa City, IA). RTE was measured by periodically applying a 2.5-mV bipolar pulse and was calculated using Ohm's law. The bathing Ringer solution was composed of 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose. Chambers were constantly gassed with a mixture of 95% O2, 5% CO2 at 37°C, which maintained the pH at 7.4. Following a 5-min equilibration period, the baseline Isc was recorded. To determine the amiloride-sensitive Isc, the sodium channel blocker amiloride was added to the apical cell chamber to a concentration of 10 µM. Subsequently, the cAMP agonist forskolin (10 µM) and then the CFTR channel blocker CFTRinh-172 (10 µM; Calbiochem, San Diego, CA; Ref. 26) were added to determine stimulated CFTR currents.

Quantitative HGF concentration measurement. The basolateral media from control, epithelial-fibroblast coculture, and FCM conditions were pooled over a 1 wk interval. This media was concentrated by centrifugal ultrafiltration with a Microcon YM-10 concentrator. HGF concentration was determined using the Quantikine human HGF immunoassay (R&D Systems) per the manufacturer's instructions.

Assessment of the effects of HGF on HBE cell phenotype. To assess the contribution of HGF to HBE cell phenotype in control and fibroblast coculture conditions, HBE cells were cultured in the presence and absence of 300 ng/ml neutralizing HGF antibody L2G7 or 10 ng/ml rhHGF for 21 days. In control conditions, isotype-matched mouse IgG2a was used at the same concentration. All antibodies and rhHGF were added to the basolateral media, which was changed twice weekly. Following 21 days of incubation, the RTE, cell density, and percentage of ciliated cells were measured as described above.

Statistics. Results are expressed as means ± SE. Significance was determined by ANOVA with Bonferroni post hoc analysis or by Student's t-test where appropriate, unless otherwise indicated. Because of inherent variability between donors, the data shown are the results obtained from one donor, unless otherwise indicated. Similar results were observed in independent experiments using three different donors.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial-fibroblast interactions promote normal epithelial differentiation. To determine whether factors secreted by subepithelial fibroblasts are sufficient to promote normal HBE differentiation, we compared the morphology of HBE cells cultured in serum-free differentiation media (2% USG) to that of HBE cocultured with subepithelial fibroblasts in 10% FBS. As previously shown (8, 9), HBE cells cultured at an air-liquid interface in USG developed into a pseudostratified columnar epithelium, with apical cilia and nuclei organized towards the basolateral surface (Fig. 1A). When the USG was replaced with 10% FBS, the HBE cells developed into a stratified squamous layer with a decreased cell density (data not shown), presumably as a result of the TGF-beta present in the serum (9, 13, 15, 24, 27). In contrast, when primary lung fibroblasts were cocultured on the undersurface of the Transwell filter, HBE cells developed into a normal polarized ciliated layer, despite the presence of serum (Fig. 1C). This suggests that subepithelial fibroblasts secrete factors that promote normal epithelial differentiation. To determine whether the epithelial-mesenchymal interaction requires that the cells are in near proximity, we cultured HBE cells in FCM, which was established by culturing fibroblasts on the bottom of the Transwell in 10% FBS. Under these conditions, in which the subepithelial mesenchyme was not adjacent to the HBE, the epithelial layer developed into a stratified squamous epithelium (Fig. 1B), similar to the phenotype seen when the HBE cells were cultured in FBS alone. These results suggest that direct epithelial-mesenchymal interactions are important for normal HBE cell differentiation.

Epithelial-fibroblast interactions promote ciliogenesis. To further characterize the effect of the subepithelial fibroblasts on HBE cell differentiation, we examined the number of ciliated cells present in epithelial-fibroblast coculture compared with standard culture (Fig. 2, A and B). HBE cells that were cocultured for 2 wk developed 16.8 ± 3.5% (n = 4; P = 0.003) more ciliated cells as measured by manual counting of random tubulin-labeled fields (Fig. 2C); this represents a 40% increase in ciliated cells compared with standard USG culture methods. As an alternate measurement of the amount of cilia, these results were confirmed by measurement of the total fluorescence generated by the cilia marker acetylated tubulin. Similar to the results obtained by manual counting, there was a 12.9 ± 3.8% (n = 4; P = 0.015) increase in the area covered by cilia as measured by image analysis of random fields (Fig. 2D). These results suggest that factors secreted by subepithelial fibroblasts either promote ciliogenesis or prolong ciliated cell survival.


Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 2. Effect of epithelial-fibroblast interactions on ciliogenesis after 2 wk in culture. Shown are confocal microscopy images of cilia in control (A) vs. coculture (B) conditions using immunocytochemistry for acetylated tubulin as a ciliary marker identified with Alexa 488 (green) (magnification x400). C: mean percent cilia in control and coculture HBE cells. Cilia were quantified by dividing the number of ciliated cells by the total cell count (%Cells, black bars) and by the percentage of pixels occupied by Alexa 488 (%Area, gray bars). Data shown are mean percentages of cilia ± SE, n = 4 filters. *Significantly different from control conditions.

 
Epithelial-fibroblast interactions accelerate epithelial wound repair. Because factors secreted by fibroblasts are known to accelerate epithelial wound repair (18), we next investigated whether epithelial-fibroblast coculture would affect HBE wound closure. Matched HBE cell cultures that were initially differentiated in USG were grown for 2 wk in 2% USG, FCM, or epithelial-fibroblast coculture. Subsequently, the epithelial layer was mechanically injured, the filters were serially fixed, and F-actin immunohistochemistry performed. Twenty hours following epithelial denudation, the scrape injury remained visibly patent in the control conditions (Fig. 3A) and was partially closed in FCM conditions (Fig. 3B). However, in the HBE cells that were cocultured with fibroblasts, the wound was completely reepithelialized at the same time point (Fig. 3C). As an additional measure of the rate of epithelial repair, wound closure was assessed by serial measurements of RTE. As expected, RTE sharply declined following epithelial injury and subsequently increased as the wound closed to levels exceeding baseline (Fig. 3D). The rate of restoration of RTE was markedly accelerated in epithelial-fibroblast cocultures compared with control (n = 4; P < 0.001; Fig. 3D). The rate of epithelial wound closure in FCM was accelerated with respect to control conditions, although not to the degree seen in fibroblast coculture, suggesting that ECM and secreted factors from the subepithelial mesenchyme are important for epithelial regeneration following injury.


Figure 3
View larger version (156K):
[in this window]
[in a new window]

  		Fig. 3. Epithelial wound repair after mechanical injury. Parallel differentiated HBE cultures were injured by mechanical scrape and allowed to heal for 24 h. Displayed are representative F-actin immunohistochemistry of HBE cultured in control (A), FCM (B), and coculture (C) conditions, imaged 20 h postinjury, magnification x200. D: serial measurement of transepithelial resistance (RTE) following mechanical scrape injury in control, FCM, and coculture conditions. Data shown are mean RTE ± SE, n = 4 filters. *Significantly different from control conditions.

 
HBE cells cultured in epithelial-fibroblast coculture exhibit diminished polarized ion transport. Based on our findings that fibroblast coculture augmented ciliogenesis and accelerated wound closure, we anticipated that the electrophysiological properties of HBE cells would also be heightened. To test this, HBE cultures were placed in Ussing chambers following 21 days of culture in 2% USG, FCM, or epithelial-fibroblast coculture. As shown in Fig. 4, HBE cultured in 2% USG generated a standard Isc tracing for normal HBE cells (Fig. 4A, top tracing). A large amiloride-sensitive (ENaC) current comprised the majority of the transepithelial current, and a smaller forskolin-stimulated CFTR current was also present. However, when cultured in FCM or in epithelial-fibroblast coculture, the magnitude of the transepithelial current was markedly diminished. Summary data derived from four independent experiments (3 non-CF HBE cell cultures and 1 CF culture) are shown in Table 2. These findings indicate that the morphological and electrophysiological phenotypes of HBE cells are dictated by different mechanisms and suggest that factors other than the underlying mesenchyme are important in regulating the ion transport properties of airway epithelium.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Effect of culture method on the electrophysiological properties of HBE cells. A: representative short-circuit current (Isc) tracings of HBE cells cultured in control, FCM, and coculture conditions. RTE is calculated using Ohm's law based on the Isc deflection induced by a 2.5-mV bipolar pulse. Subsequently, 10 µM amiloride was added to the apical surface, and the decrement in Isc is reflective of the epithelial sodium channel (ENaC) current. To determine the stimulated CFTR current, Cl– conductance is stimulated by 10 µM forskolin, and 10 µM CFTRinh-172 is subsequently added apically to block CFTR. The magnitude of the change in Isc is reflective of CFTR current. B: effect of hydrocortisone on vectorial ion transport. Parallel HBE cells were cultured in either control or coculture conditions ± 0.1 µM hydrocortisone for 14 days. Data shown are mean PD ± SE, n = 6 filters. *Significant difference from HBE cells cultured without hydrocortisone. PD, potential difference.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Electrophysiological effects of epithelial-fibroblast coculture

 
Next, we tested whether additives to the basolateral media could increase polarized current in the coculture system. In standard HBE cell differentiation media, hydrocortisone is typically added to serum-free HBE differentiation media to augment ENaC current (41). Therefore, we added 0.1 µM hydrocortisone to the basolateral media (Fig. 4B). This had no effect on the PD in the control conditions (P = 0.19; n = 6), suggesting that there is sufficient glucocorticoid in USG to stimulate ENaC. However, hydrocortisone more than doubled the PD in the cocultures (P = 0.0001; n = 6). Therefore, the reduced polarized ion transport in this epithelial-fibroblast coculture system can be augmented with the addition of physiological regulators of ENaC activity.

HGF from subepithelial fibroblasts contributes to HBE cell differentiation. Because HGF is secreted from fibroblasts (33) and the HGF receptor (c-Met) is expressed on the basolateral surface of HBE (42), we reasoned that HGF secreted by subepithelial fibroblasts may be a critical growth factor to support normal HBE differentiation. Furthermore, HGF has been shown to increase ciliogenesis (42), decrease the amiloride-sensitive Isc (43), and counteract the effects of TGF-beta (46) in a manner similar to what was observed in epithelial-fibroblast coculture. To determine the amount of HGF secreted by subepithelial fibroblasts, ELISA was performed on the basolateral media from control, coculture, and FCM. As shown in Fig. 5A, HGF is present in the fibroblast containing cultures in sufficient concentrations to act as a mitogen and stimulate ciliogenesis in HBE (33, 34, 42). In control conditions and media alone, minimal amounts of HGF were present. These results confirm that subepithelial fibroblasts secrete HGF in sufficient concentrations to support epithelial differentiation.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. Effect of hepatocyte growth factor (HGF) on HBE cell phenotype. A: HGF concentration in the basolateral media in the different culture conditions. *Significantly different from control conditions as determined by Kruskal-Wallis ANOVA on ranks with Dunn's post hoc analysis. B–D: HBE cells were cultured in control and coculture conditions for 21 days with 300 ng/ml HGF neutralizing antibody (Ab) L2G7, 10 ng/ml recombinant human HGF (rhHGF), or 300 ng/ml control Ab. *Significantly different from cells cultured under the same conditions in the presence of control Ab. B: effect of HGF on RTE in control and coculture conditions. C: effect of HGF on cell density in control and coculture conditions. D: effect of HGF on ciliogenesis in control and coculture conditions. Data shown are means ± SE, n = 4 filters.

 
To assess the contribution of HGF derived from the subepithelial mesenchyme on HBE cell phenotype, we treated control and epithelial-fibroblast cultures with an HGF-neutralizing antibody or an isotype-matched control antibody for 21 days prior to measurement of RTE, cell density, and cilia density (Fig. 5, B–D). Neutralization of HGF with 300 ng/ml neutralizing antibody L2G7 markedly decreased the RTE, cell density, and percentage of ciliated cells in coculture conditions. However, HGF neutralization had no effect on the HBE cell phenotype when the cells were cultured in USG. These results indicate that HGF derived from the subepithelial fibroblasts is a critical factor in the development of a normal HBE phenotype in serum-containing media. Next, as shown in Fig. 5, B–D, we assessed the effects of recombinant HGF on HBE cells cultured in control and epithelial-fibroblast coculture conditions. Whereas 10 ng/ml rhHGF did not affect the RTE, rhHGF increased the cell density and amount of cilia present in both control and coculture conditions. These results are in agreement with previous reports that HGF is important in ciliogenesis and acts as a mitogen for HBE (33, 34, 42). Thus subepithelial mesenchymal cells elaborate HGF, which is an important mediator for normal epithelial differentiation in our coculture system.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Despite the importance of epithelial-mesenchymal interactions in lung development and epithelial repair, little is known about how epithelial cells and fibroblasts interact in vitro. Using primary cultures of HBE cells and human lung fibroblasts, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype, despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) HGF is involved in subepithelial fibroblast modulation of HBE cell differentiation. These results indicate that the local environment established by subepithelial mesenchymal cells promotes HBE cellular differentiation and augments epithelial restoration following injury in the airways.

Although the HGF concentration was similar in coculture and FCM conditions, the subepithelial fibroblasts were only capable of promoting a well-differentiated HBE layer when the two cell types were in close proximity. We hypothesize that the local concentration of HGF and potentially other growth factors is increased when the subepithelial fibroblasts are in close proximity to the overlying HBE. Other mediators with shorter half-lives may also be secreted by the epithelium and fibroblasts and have trivial concentrations in the FCM conditions. Alternatively, the subepithelial fibroblasts may provide a barrier that isolates the HBE from the effects of TGF-beta and other serum components that promote squamous differentiation of HBE cells in the absence of anatomically positioned fibroblasts. However, the fibroblast layer did not significantly alter the electrical resistance across the Transwell filter (Table 2 and data not show), suggesting that the fibroblasts constitute a weak physical barrier. More likely is that the matrix secreted by fibroblasts alters the access of TGF-beta or other serum components to the epithelial cells. Further studies of the coculture matrix will likely be informative.

Unexpectedly, whereas epithelial-fibroblast coculture promoted normal epithelial morphological differentiation, we found that HBE cells cultured in this model developed minimal polarized ion transport. Indeed, these electrophysiological properties can be partially restored in coculture conditions with the addition of hydrocortisone (Fig. 4B) or in the presence of USG (53). Because cultured HBE cells are able to regulate ASL volume, it is believed that regulation of ion transport is dictated by autocrine mechanisms inherent to HBE cells. Since epithelial-fibroblast coculture was sufficient to promote a morphologically normal epithelial layer, the paucity of vectorial ion transport in coculture suggests that alternative mechanisms exist in vivo to regulate ion transport. We speculate that these nonautocrine factors are important for ENaC and CFTR channel expression in HBE cells but may not necessarily be involved in the physiological regulation of the activity of these channels.

There has recently been a resurgence of investigation into the plasticity of airway epithelium. In vivo, basal bronchial epithelial cells are capable of assuming ciliated, squamous, columnar, and goblet cell phenotypes following injury (1). In a recent murine model reported by Tyner et al. (45), the development of epithelial hyperplasia and mucus metaplasia in the airway involved transdifferentiation of ciliated cells to goblet cells. Additionally, epithelial-to-mesenchymal transition has been demonstrated in lung epithelium, at least in animal models (14, 52, 54, 55), and is believed to be involved in the pathogenesis of lung cancer (16), obliterative bronchiolitis (49), and idiopathic pulmonary fibrosis (52). Our findings provide additional laboratory evidence of epithelial cell plasticity, and demonstrate that the morphological and electrophysiological phenotypes of HBE cells are not intimately linked. Airway cell differentiation into a ciliated epithelium may be necessary for normal mucociliary clearance, however, ciliary differentiation is not sufficient for establishment of significant chloride and sodium conductances. Hence the ion transport properties of airway epithelium appear regulated by additional mechanisms.

In summary, these data suggest that secretion of HGF from subepithelial mesenchymal cells is important for the preservation of epithelial integrity and mucociliary clearance in the airways. Traditionally, subepithelial fibroblast hyperplasia is regarded as a pathological feature of airway remodeling and believed to contribute to airway luminal narrowing in obstructive airway diseases. Furthermore, heightened levels of HGF are known to promote bronchial neoplasia (35, 40, 44, 50). Because subepithelial fibroblasts elaborate factors that increase the rate of wound closure and augment ciliogenesis, we speculate that subepithelial fibroblast proliferation following epithelial injury may have a beneficial role in airway epithelial repair.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by an Institutional National Research Service award (M. Myerburg), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-56490 (J. M. Pilewski), National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-050840 (C. A. Feghali-Bostwick), the American Lung Association of Pennsylvania (J. M. Pilewski), and a Cystic Fibrosis Research Development Program grant to the University of Pittsburgh (J. M. Pilewski).


   ACKNOWLEDGMENTS
 
We thank Galaxy Biotech for providing HGF neutralizing antibody L2G7 and isotype-matched control antibody. Additionally, we thank Dr. Kenneth McCurry and the Lung Transplant Program at the University of Pittsburgh Medical Center for facilitating tissue acquisition.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Pilewski, Division of PACCM, Univ. of Pittsburgh Medical Center, 628 NW Montefiore Univ. Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213 (e-mail: pilewskijm{at}upmc.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Basbaum C, Jany B. Plasticity in the airway epithelium. Am J Physiol Lung Cell Mol Physiol 259: L38–L46, 1990.[Abstract/Free Full Text]
  2. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 167: 1360–1368, 2003.[Abstract/Free Full Text]
  3. Butterworth MB, Edinger RS, Johnson JP, Frizzell RA. Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool. J Gen Physiol 125: 81–101, 2005.[Web of Science][Medline]
  4. Davenport EA, Nettesheim P. Regulation of mucociliary differentiation of rat tracheal epithelial cells by type I collagen gel substratum. Am J Respir Cell Mol Biol 14: 19–26, 1996.[Abstract]
  5. Devor DC, Bridges RJ, Pilewski JM. Pharmacological modulation of ion transport across wild-type and {Delta}F508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell Physiol 279: C461–C479, 2000.[Abstract/Free Full Text]
  6. Erjefalt JS, Erjefalt I, Sundler F, Persson CG. In vivo restitution of airway epithelium. Cell Tissue Res 281: 305–316, 1995.[Web of Science][Medline]
  7. Gray T, Koo JS, Nettesheim P. Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium. Toxicology 160: 35–46, 2001.[CrossRef][Web of Science][Medline]
  8. Gray TE, Guzman K, Davis CW, Abdullah LH, Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14: 104–112, 1996.[Abstract]
  9. Gruenert DC, Finkbeiner WE, Widdicombe JH. Culture and transformation of human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 268: L347–L360, 1995.[Abstract/Free Full Text]
  10. Hogan BL. Morphogenesis. Cell 96: 225–233, 1999.[CrossRef][Web of Science][Medline]
  11. Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM, Lordan JL. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 105: 193–204, 2000.[CrossRef][Web of Science][Medline]
  12. Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Glucocorticoid-stimulated lung epithelial Na+ transport is associated with regulated ENaC and sgk1 expression. Am J Physiol Lung Cell Mol Physiol 282: L631–L641, 2002.[Abstract/Free Full Text]
  13. Jetten AM. Growth and differentiation factors in tracheobronchial epithelium. Am J Physiol Lung Cell Mol Physiol 260: L361–L373, 1991.[Abstract/Free Full Text]
  14. Kasai H, Allen JT, Mason RM, Kamimura T, Zhang Z. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res 6: 56, 2005.[CrossRef][Medline]
  15. Ke Y, Gerwin BI, Ruskie SE, Pfeifer AM, Harris CC, Lechner JF. Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents. Am J Pathol 137: 833–843, 1990.[Abstract]
  16. Kiemer AK, Takeuchi K, Quinlan MP. Identification of genes involved in epithelial-mesenchymal transition and tumor progression. Oncogene 20: 6679–6688, 2001.[CrossRef][Web of Science][Medline]
  17. Kim KJ, Wang L, Su YC, Gillespie GY, Salhotra A, Lal B, Laterra J. Systemic anti-hepatocyte growth factor monoclonal antibody therapy induces the regression of intracranial glioma xenografts. Clin Cancer Res 12: 1292–1298, 2006.[Abstract/Free Full Text]
  18. Knight D. Epithelium-fibroblast interactions in response to airway inflammation. Immunol Cell Biol 79: 160–164, 2001.[CrossRef][Medline]
  19. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109: 571–577, 2002.[CrossRef][Web of Science][Medline]
  20. Knowles MR, Buntin WH, Bromberg PA, Gatzy JT, Boucher RC. Measurements of transepithelial electric potential differences in the trachea and bronchi of human subjects in vivo. Am Rev Respir Dis 126: 108–112, 1982.[Web of Science][Medline]
  21. Krein PM, Sabatini PJ, Tinmouth W, Green FH, Winston BW. Localization of insulin-like growth factor-I in lung tissues of patients with fibroproliferative acute respiratory distress syndrome. Am J Respir Crit Care Med 167: 83–90, 2003.[Abstract/Free Full Text]
  22. Krein PM, Winston BW. Roles for insulin-like growth factor I and transforming growth factor-beta in fibrotic lung disease. Chest 122: 289S-293S, 2002.[CrossRef][Web of Science][Medline]
  23. Kreindler JL, Jackson AD, Kemp PA, Bridges RJ, Danahay H. Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract. Am J Physiol Lung Cell Mol Physiol 288: L894–L902, 2005.[Abstract/Free Full Text]
  24. Lechner JF, Haugen A, McClendon IA, Shamsuddin AM. Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum. Differentiation 25: 229–237, 1984.[CrossRef][Web of Science][Medline]
  25. Limat A, Mauri D, Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells. J Invest Dermatol 107: 128–135, 1996.[CrossRef][Web of Science][Medline]
  26. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, Verkman AS. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110: 1651–1658, 2002.[CrossRef][Web of Science][Medline]
  27. Masui T, Wakefield LM, Lechner JF, LaVeck MA, Sporn MB, Harris CC. Type beta transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc Natl Acad Sci USA 83: 2438–2442, 1986.[Abstract/Free Full Text]
  28. Matsui H, Davis CW, Tarran R, Boucher RC. Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia. J Clin Invest 105: 1419–1427, 2000.[Web of Science][Medline]
  29. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005–1015, 1998.[CrossRef][Web of Science][Medline]
  30. Matsui H, Randell SH, Peretti SW, Davis CW, Boucher RC. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J Clin Invest 102: 1125–1131, 1998.[Web of Science][Medline]
  31. Mutsaers SE, Bishop JE, McGrouther G, Laurent GJ. Mechanisms of tissue repair: from wound healing to fibrosis. Int J Biochem Cell Biol 29: 5–17, 1997.[CrossRef][Web of Science][Medline]
  32. Myerburg MM, Butterworth MB, McKenna EE, Peters KW, Frizzell RA, Kleyman TR, Pilewski JM. Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hypersabsorption in cystic fibrosis. J Biol Chem 281: 27942–27949, 2006.[Abstract/Free Full Text]
  33. Ohmichi H, Koshimizu U, Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development. Development 125: 1315–1324, 1998.[Abstract]
  34. Ohmichi H, Matsumoto K, Nakamura T. In vivo mitogenic action of HGF on lung epithelial cells: pulmotrophic role in lung regeneration. Am J Physiol Lung Cell Mol Physiol 270: L1031–L1039, 1996.[Abstract/Free Full Text]
  35. Pilewski JM, Bumbalo TS 3rd, Davis AG, Siegfried JM. Hepatocyte growth factor promotes tumor growth in a novel in vivo model of human lung cancer. Am J Respir Cell Mol Biol 24: 556–562, 2001.[Abstract/Free Full Text]
  36. Pilewski JM, Liu L, Henry AC, Knauer AV, Feghali-Bostwick CA. Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition. Am J Pathol 166: 399–407, 2005.[Abstract/Free Full Text]
  37. Pitkanen OM, Tanswell AK, O'Brodovich HM. Fetal lung cell-derived matrix alters distal lung epithelial ion transport. Am J Physiol Lung Cell Mol Physiol 268: L762–L771, 1995.[Abstract/Free Full Text]
  38. Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160: S12–S16, 1999.[Abstract/Free Full Text]
  39. Roche WR. Fibroblasts and asthma. Clin Exp Allergy 21: 545–548, 1991.[CrossRef][Web of Science][Medline]
  40. Rong S, Bodescot M, Blair D, Dunn J, Nakamura T, Mizuno K, Park M, Chan A, Aaronson S, Vande Woude GF. Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor. Mol Cell Biol 12: 5152–5158, 1992.[Abstract/Free Full Text]
  41. Sachs LA, Finkbeiner WE, Widdicombe JH. Effects of media on differentiation of cultured human tracheal epithelium. In Vitro Cell Dev Biol Anim 39: 56–62, 2003.[CrossRef]
  42. Shen BQ, Panos RJ, Hansen-Guzman K, Widdicombe JH, Mrsny RJ. Hepatocyte growth factor stimulates the differentiation of human tracheal epithelia in vitro. Am J Physiol Lung Cell Mol Physiol 272: L1115–L1120, 1997.[Abstract/Free Full Text]
  43. Shen BQ, Widdicomb JH, Mrsny RJ. Hepatocyte growth factor inhibits amiloride-sensitive Na+ channel function in cystic fibrosis airway epithelium in vitro. Pulm Pharmacol Ther 12: 157–164, 1999.[CrossRef][Web of Science][Medline]
  44. Stabile LP, Lyker JS, Land SR, Dacic S, Zamboni BA, Siegfried JM. Transgenic mice overexpressing hepatocyte growth factor in the airways show increased susceptibility to lung cancer. Carcinogenesis 27: 1547–1555, 2006.[Abstract/Free Full Text]
  45. Tyner JW, Kim EY, Ide K, Pelletier MR, Roswit WT, Morton JD, Battaile JT, Patel AC, Patterson GA, Castro M, Spoor MS, You Y, Brody SL, Holtzman MJ. Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. J Clin Invest 116: 309–321, 2006.[CrossRef][Web of Science][Medline]
  46. Ueki T, Kaneda Y, Tsutsui H, Nakanishi K, Sawa Y, Morishita R, Matsumoto K, Nakamura T, Takahashi H, Okamoto E, Fujimoto J. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 5: 226–230, 1999.[CrossRef][Web of Science][Medline]
  47. Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA, Zabner J, Welsh MJ. Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature 422: 322–326, 2003.[CrossRef][Medline]
  48. Vermeer PD, Panko L, Karp P, Lee JH, Zabner J. Differentiation of human airway epithelia is dependent on erbB2. Am J Physiol Lung Cell Mol Physiol 291: L175–L180, 2006.[Abstract/Free Full Text]
  49. Ward C, Forrest IA, Murphy DM, Johnson GE, Robertson H, Cawston TE, Fisher AJ, Dark JH, Lordan JL, Kirby JA, Corris PA. Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients. Thorax 60: 865–871, 2005.[Abstract/Free Full Text]
  50. Weidner KM, Behrens J, Vandekerckhove J, Birchmeier W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol 111: 2097–2108, 1990.[Abstract/Free Full Text]
  51. Whitsett JA. Intrinsic and innate defenses in the lung: intersection of pathways regulating lung morphogenesis, host defense, and repair. J Clin Invest 109: 565–569, 2002.[CrossRef][Web of Science][Medline]
  52. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, Borok Z. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 166: 1321–1332, 2005.[Abstract/Free Full Text]
  53. Wiszniewski L, Jornot L, Dudez T, Pagano A, Rochat T, Lacroix JS, Suter S, Chanson M. Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis. Am J Respir Cell Mol Biol 34: 39–48, 2006.[Abstract/Free Full Text]
  54. Xu YD, Hua J, Mui A, O'Connor R, Grotendorst G, Khalil N. Release of biologically active TGF-beta1 by alveolar epithelial cells results in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 285: L527–L539, 2003.[Abstract/Free Full Text]
  55. Yao HW, Xie QM, Chen JQ, Deng YM, Tang HF. TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro. Life Sci 76: 29–37, 2004.[CrossRef][Web of Science][Medline]
  56. Yoon JH, Gray T, Guzman K, Koo JS, Nettesheim P. Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. Am J Respir Cell Mol Biol 16: 724–731, 1997.[Abstract]
  57. Zabner J, Freimuth P, Puga A, Fabrega A, Welsh MJ. Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J Clin Invest 100: 1144–1149, 1997.[Web of Science][Medline]
  58. Zahm JM, Debordeaux C, Raby B, Klossek JM, Bonnet N, Puchelle E. Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium. J Cell Physiol 185: 447–453, 2000.[CrossRef][Web of Science][Medline]
  59. Zhang S, Smartt H, Holgate ST, Roche WR. Growth factors secreted by bronchial epithelial cells control myofibroblast proliferation: an in vitro co-culture model of airway remodeling in asthma. Lab Invest 79: 395–405, 1999.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
K. L. Veraldi, B. T. Gibson, H. Yasuoka, M. M. Myerburg, E. A. Kelly, S. Balzar, N. N. Jarjour, J. M. Pilewski, S. E. Wenzel, and C. A. Feghali-Bostwick
Role of Insulin-like Growth Factor Binding Protein-3 in Allergic Airway Remodeling
Am. J. Respir. Crit. Care Med., October 1, 2009; 180(7): 611 - 617.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. M. Myerburg, E. E. McKenna, C. J. Luke, R. A. Frizzell, T. R. Kleyman, and J. M. Pilewski
Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis
Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L932 - L941.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/L1352    most recent
00328.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Myerburg, M. M.
Right arrow Articles by Pilewski, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Myerburg, M. M.
Right arrow Articles by Pilewski, J. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.