help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on December 12, 2003, doi:10.1165/rcmb.2003-0145OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0145OCv1
30/6/784    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwarz, M. A.
Right arrow Articles by Lee, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwarz, M. A.
Right arrow Articles by Lee, M. K.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 784-792, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0145OC

Epithelial–Mesenchymal Interactions Are Linked to Neovascularization

Margaret A. Schwarz, ZeSheng Wan, Jie Liu and Matthew K. Lee

Departments of Pediatrics, Cardiothoracic Surgical Research and Surgery, Childrens Hospital Los Angeles Research Institute, Los Angeles; Center for Craniofacial Molecular Biology, and University of Southern California Keck School of Medicine and School of Dentistry, Los Angeles, California; and Departments of Surgery and Pediatrics, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey

Address correspondence to: Dr. Margaret Schwarz, UMDNJ-Robert Wood Johnson Medical School, 125 Paterson Street, CAB 7036, New Brunswick, NJ 08903. E-mail: m.schwarz{at}umdnj.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung morphogenesis is dependent on interactions between mesenchymal and epithelial cells. We have previously demonstrated that inhibition of neovascularization by endothelial monocyte–activating polypeptide (EMAP) II also attenuates fetal lung morphogenesis in vivo, and hypothesized that epithelial–mesenchymal interactions are regulated by vascular signals. To address this postulate, we evaluated the formation of epithelial cysts in vitro and assessed this complex interaction through: (i) identification of vascular formation in vitro; (ii) assessment of the effect of selective vascular inhibition on cell viability, proliferation, and cellular interactions as measured by epithelial cyst formation; and (iii) examination of whether there is an interdependent relationship between epithelial and mesenchymal cells and a vascular mediator's protein expression. Vascular networks in vitro formed in direct relationship to the presence of epithelial cysts. Disruption of the vasculature by delivery of a selective antiangiogenic protein EMAP II was associated with disruption of epithelial cyst formation. Lastly, control of the vascular formation regulatory protein EMAP II is a direct result of epithelial–mesenchymal cell interactions. These findings suggest that vascular formation modulates and is modulated by the normal cellular communication and interactions that direct lung morphology.

Abbreviations: endothelial-monocyte–activating polypeptide, EMAP • mature 21-kD form of EMAP, mEMAP II • phosphate-buffered saline, PBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung morphogenesis, including epithelial differentiation, is a complex series of instructive and permissive interactions between the epithelium and mesenchyme (1). This direct communication between the immature epithelium and rudimentary mesenchyme promotes cellular proliferation, branching, and neovascularization (2). Interruption of this process leads to impaired lung formation. A classic example of this principle is found in epithelial cell differentiation. Distal fetal lung epithelium and proximal mesenchymal recombinants determined that epithelial differentiation can be regulated by the mesenchyme in a position-specific manner (3). Similarly, endothelial cell maturation of totipotent extraembryonic mesodermal is dependent on their environment, as exemplified by vascular endothelial growth factor promotion of pluripotent flk-1 cells to differentiate into an endothelial cell. In contrast, platelet-derived growth factor-BB promotes smooth muscle cells and pericytes differentiation of this pluripotent cell (4, 5). Thus terminal differentiation of a pluripotent cell within the developing lung may therefore be determined by its signaling microenvironment.

Given this context, it is highly plausible that the evolving vascular compartment also represents a distinct cell population with significant regulatory contributions to lung development. Multiple reports have suggested that neovascular regulators concomitantly modulate lung morphogenesis (6–9). These findings suggest that vascular cell populations may contribute to the regulation of distal airspace development. However, little is known regarding the mechanisms mediating this regulation. We hypothesized that inhibition of vascular formation would disrupt the ability of epithelial and mesenchymal cells to organize into alveolar structures. To address this postulate, we sought to: (i) identify whether neovascular development occurs during epithelial cyst formation in vitro; (ii) determine if disrupted neovascularization attenuates the organization of epithelial and mesenchymal cells into epithelial cysts in vitro; and (iii) identify whether the interaction between epithelial and mesenchymal cells is associated with modulated expression of vascular regulators.

One such regulator, endothelial-monocyte activating polypeptide (EMAP) II, is processed by unknown mechanisms to a mature 21-kD form (mEMAP II) that functions as a potent anti-angiogenic peptide capable of inducing migrating and proliferating endothelial cells to undergo apoptosis (10, 11). Our original observations suggested that EMAP II was an ideal candidate to examine vascular–epithelial/mesenchymal interactions because EMAP II: (i) was highly expressed during the less vascularized period of the pseudoglandular stage; (ii) was localized to the epithelial–mesenchymal junction before its marked downregulation on entering the canalicular stage; (iii) expression was limited to a perivascular distribution during the ensuing "vascular" stage (similar to that seen in the adult) (12); and (iv) exogenous delivery of mEMAP II attenuates distal fetal lung airspace formation as well as neovascularization in a fetal lung allograft model (7).

Although culture of individual cellular components of the fetal lung is readily feasible, the microenvironment of the three-dimensional culture is more relevant to an in vivo environment (13). We exploited the ability of freshly isolated dissociated fetal mouse lung epithelial and mesenchymal cells to reaggregate and organize into cystic structures in vitro serving as a proxy of alveolar architectural development. This pattern of recombination offered a unique advantage as fetal cells sort themselves into epithelial and mesenchymal components, polarize, produce a basement membrane, and branch in a pattern resembling the tissue of origin (14). Integration of this alveolar development model allowed us to analyze in vitro the role of neovascularization in the formation of the epithelial cyst. Consistent with in vivo lung formation, fetal lung recombinants formed epithelial cysts surrounded by vascular cells. Corresponding with organotypic formation, defined as reaggregation of epithelial components surrounded by mesenchyme, vascular networks were present as early as 24 h after isolation and plating. Consistent with normal parallel formation of the bronchi and vasculature, vascular networks formed in direct relationship to the presence of epithelial cysts surrounded by mesenchyme. Disruption of the vasculature by delivery of the selective anti-angiogenic protein mEMAP II was associated with attenuated epithelial cyst formation. Lastly, EMAP II's protein expression is regulated by epithelial–mesenchymal cell interactions. These findings suggest that a vascular compartment arises from epithelial–mesenchymal recombination, and that this compartment is important to the formation of epithelial cysts. We speculate that the contribution of neovascular precursors to cyst formation in vitro recapitulates a similar role during the fetal lung morphogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Isolation and Culture
Fetal epithelial cells were isolated from timed pregnant CD-1 pathogen-free mice housed and handled according to the animal care committee. At Day 15–17 dpc (days post-coital) during the late pseudoglandular through the canalicular stage, dams were humanely killed, fetuses removed via C-section, and the lungs dissected out as a block. The fetal lungs were then placed in iced phosphate-buffered saline (PBS), rinsed, and minced finely into 1 mm3 in the presence of DNase (6,000 U/ml). Following pelleting of the cells at 420 g, the pellets were resuspended in warm 0.02% trypsin in PBS and DNase (1 ml/fetus) and gently shaken at 37°C in a T75 flask. The digested tissue was filtered through a 100-µm nylon cell strainer (420 g for 5 min). Epithelial cells were then isolated using differential adherence techniques (15, 16). On final plating, cells were placed in either 30-mm dishes for protein analysis or 96-well plates for 3H thymidine experiments. Cell populations were used for assays only when they displayed >= 85% epithelial cells.

Mesenchymal cells were isolated using differential plating (17) from timed pregnant CD-1 pathogen-free mice housed and handled according to the animal care committee. At Day 14 dpc, isolated fetal lungs were minced and dissociated in the presence of PBS and 0.3% trypsin and 0.1% EDTA for 2 min. A single-cell suspension was obtained by forcing cell aggregates and pieces of tissue through a micropipet several times. Cells were filtered through a 100-µm nylon strainer and resuspended in minimal essential medium with 10% fetal calf serum, nonessential amino acids, 0.29 mg/ml L-glutamine, 100 U penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B for 30 min at 37°C to allow the mesenchymal cells to attach. After the initial incubation, the nonattached cells were removed and the attached mesenchymal cells were cultured in medium containing 10% fetal calf serum in confluent and subconfluent conditions. On plating, cells were placed in either 30-mm dishes for protein analysis or 96-well plates for 3H thymidine experiments.

Co-Culture
Organotypic murine lung cultures were performed following the protocol of Schuger and coworkers (18–20). In brief, we initially identified those 15 dpc embryos that have the flk-1 LacZ gene. The embryo was numbered, a piece of the tail of the embryo removed for ß galactosidase, and simultaneously as the assay was being performed over the next hour, the lungs were removed en bloc and placed in ice-cold PBS. Once the positive lungs were identified (as noted by corresponding tail numbers that were positive for X-gal), the cells dissociated in PBS containing 0.3% trypsin and 0.1% EDTA for 10 min at 37°C before being filtered through a 100-µm pore mesh. The dissociated cell mixture was then resuspended in minimal essential medium (Gibco-BRL, Gaithersburg, MD) with nonessential amino acids and plated at a concentration of 2–2.5 x 106 cells/ml in uncoated 8-well chamber slides. Experiments were performed in the presence of vehicle, mEMAP II (mature 0.8–3.2 µg/ml), EMAP II peptide antibody (3–6 µg/ml), and rabbit IgG (control). Epithelial cyst formation was evaluated by counting the number of epithelial cysts per high-power field (HPF). We analyzed 10 fields per condition (n = 8/group performed on four different occasions).

3H Thymidine Studies
Cells were plated in 96-well plates and exposed to vehicle (medium) or EMAP II (3.5µg/ml). Sixteen hours before harvesting, 10 µCi/ml 3H thymidine was added to each well. Cells were harvested for 3H counts at 24, 48, and 72 h following exposure to either vehicle or mEMAP II (n = 2–3/group performed on three different occasions).

Protein Isolation and Western Blotting
Cells were lysed in 50 mM Tris pH 7.4, 0.9 N NaCl, 1% NP-40, and 0.01% NaN3, in the presence of the protease inhibitors (aprotinin 20 µg/ml, leupeptin 20 µg/ml, and pepstatin A 20 µg/ml), and stored at –70°C. For Western analysis, homogenates were cleared by centrifugation at 14,000 x g for 20 min, the protein concentration determined by Bradford analysis (Bio-Rad, Hercules, CA), and the samples normalized by protein content. Equal amounts of protein was then electrophoresed on a 12% SDS-PAGE gel, transferred to Immobilon-P, blocked overnight in a casein-based blocking solution (Boehringer-Mannheim, Indianapolis, IN), and probed with a rabbit anti-EMAP II antibody (21), keratin (Sigma, St. Louis, MO), ß-galactosidase (Caltag, Burlingame, CA), PCNA (Sigma), laminin, and fibronectin (both from Santa Cruz, Santa Cruz, CA). Specific binding was detected using a chemiluminescence substrate (Pierce, Rockford, IL) and XAR-5 film (Eastman Kodak, Rochester, NY). Quantitative analysis was accomplished using a Molecular Dynamics Personal Densitometer (Amersham Biosciences, Piscataway, NJ) and samples were normalized for background (n = 3/group performed on three different occasions).

Immunohistochemistry Analysis
As previously described (21), cells in the eight-well dish were fixed in 4% paraformaldehyde and dehydrated. We used a peptide generated polyclonal antibody of EMAP II (1µg/ml). Using a histostain kit from Zymed (San Francisco, CA), after blocking, the sections were exposed to the primary antibody overnight at 4°C. Cells were then incubated with secondary biotinylated antibody as per the manufacturer's protocol. A brief incubation with the Streptavidin-HRP conjugate system (Zymed) was followed by development using the chromogen substrate aminoethylcarbazole (AEC) (n = 2/group performed on three different occasions).

RNA In Situ Hybridization Using Dig-Labeled cRNA Probes
As we have previously described (21), the Dig RNA probe antisense and sense (control) were made using the Dig RNA Labeling Kit (SP6/T7) from Boehringer Mannheim (Indianapolis, IN). RNA in situ hybridization was performed on 5-mm paraffin-embedded material sections according to nonradioactive in situ hybridization application manual (Boehringer Mannheim). Using DEPC-treated equipment and solutions, eight-well slides underwent rehydration and incubation in a prewarmed 5 µg/ml proteinase K solution. Slides were then reimmersed in 4% PFA, treated with a 0.25% acetic anhydride, and dehydrated. Sections were exposed to a hybridization solution containing the Dig-labeled RNA probe at 50°C overnight. Slides were washed at 55°C for 30 min before being incubated with RNase A (20 µg/ml) for 30 min at 37°C. After being rinsed with 2 x SSC and Dig Nucleic Acid detection was accomplished using the Genius 3 kit from Boehringer Mannheim. Following blocking, slides were incubated with anti-Dig–AP conjugate at 4°C overnight, rinsed, and incubated with a dilute NBT/BCIP solution for 3 h at room temperature. Slides were then rinsed in water, air–dried, and mounted. Hybridization with sense probe or without probe was performed as negative control and they always showed no signals (n = 2/group performed on two different occasions). All sections were examined and photographed under light microscopy.

TUNEL Analysis of Co-Cultures
Co-cultured cells were exposed to vehicle, EMAP II (3.2 µg/ml), EMAP II antibody (6 µg/ml), or rabbit IgG. Cells were evaluated on Days 1–3 for apoptosis. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and exposed to the TUNEL reaction (containing terminal deoxynucleotidyl transferase and a nucleotide mixture in a reaction buffer). Cells were exposed to a fluorescein-conjugated antibody, counterstained with propidium iodine (0.05 µg/ml), mounted using PBS/glycerol, and observed under a fluorescent microscope (Olympus BX40; Olympus, Sunnyvale, CA) at an excitation wavelength of 488 nm and detection at 515–565 nm (n = 3–5/group performed on three different occasions) (paired t test, n = 6 on three occasions, represents the results of 10 high-power fields per n, cyst deemed to be apoptotic).

Additional co-cultures were performed using flk-1 LacZ transgenic mice. In these cultures, ß-galactosidase was revealed following fixation in 4% paraformaldehyde, where co-cultures were exposed to an X-gal solution (potassium ferricyanide, potassium ferrocyanide, MgCl2, and X-gal in a PBS solution) for 1 h at 37°C prior to rinsing and either mounting, costaining with fast red, or performing TUNEL reaction as described above (n = 2/group performed on three different occasions).

Statistics
Statistical analysis was performed using Student's t test and one-way ANOVA on the computer program Statview.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Aggregation of Freshly Dissociated Fetal Lung Cells into Cystic Structures is Associated with the Development of Reticular Networks of Endothelial Cells
The aggregation of recombined freshly isolated dissociated fetal lung cells into cystic structures in vitro has been previously characterized (17, 18, 20, 22). Dissociated fetal lung cells isolated from E (embryonic day) 15.5 mice bearing a gfp expression cassette under the control of an endothelial specific Tie-2 promoter were used to determine endothelial cell presence in the dissociated cell population. Flow cytometry analysis using 488 nm on the dissociated cell population determined that ~ 2.8% of the cells were gfp-positive (Figure 1A). Observation of the collected positive cells under flourescence microscopy confirmed a 99% gfp-positive cell population (data not shown). This suggests survival of some vascular derivatives during fetal lung dissociation. However, it is unclear if endothelial cells survive the isolation process and reaggregate into vascular networks. To address this issue, fetal lung tissue was isolated from mice bearing a LacZ expression cassette under the control of the flk-1 promoter. Flk-1 is uniquely expressed in vascular cell populations, and expression of ß-galactosidase in these cells indicates commitment to a vascular phenotype. E 15.5 dissociated fetal lungs spontaneously recombined in a three-dimensional fashion into epithelial cyst surrounded by mesenchyme as previously described (17, 18, 20, 22). When assessed for ß-galactosidase activity, a time dependent formation of a reticular network of LacZ-expressing cells was observed in association with epithelial cysts. These networks developed among the mesenchymal cells surrounding the epithelial cysts and were one cell wide (Figure 1B, Day 1; 1C, Day 2; 1D, Day 3). Monocultures of mesenchymal cells or epithelial cells did not form epithelial cysts, and did not develop reticular networks of LacZ-expressing cells. Mesenchymal cell cultures did develop well-demarcated areas of relatively dense LacZ expression, but these were never observed to assume a reticular phenotype (data not shown). Thus, the development of a reticular network of LacZ-expressing cells was uniquely associated with epithelial cyst formation.



View larger version (80K):
[in this window]
[in a new window]
  		Figure 1. Endothelial cells aggregate to form a reticular vascular network in co-culture. Dissociated fetal lung cells from 15.5 mice bearing a gfp Tie-2 promoter analyzed by flow cytometry (488 nm) indicates that 2.8% of the cell population are endothelial cells (A). Dissociated lungs from mice positive for ß-galactosidase flk-1 promoter indicated that there was an active time-dependent formation of an endothelial reticular network. This was demonstrated by vascular network progression revealed by LacZ staining on Day 1 (B), Day 2 (C), and Day 3 (D). Bar = 500 µm.

 
mEMAP II Attenuates the Formation of Endothelial Cell Networks In Vitro
Spontaneous recombination of dissociated fetal lung cells and maintenance in culture results in the development of endothelial cell structures. To determine if these structures are subject to regulation by the angiogenic inhibitor mEMAP II, cultures were exposed to mEMAP II (0.8–3.2 µg/ml). Over 72 h in culture, exogenous mEMAP II markedly reduced the relative number of endothelial cells and the reticular networks in mixed cell–type cultures (Figures 2C and 2D) relative to control cultures (Figures 2A and 2B). This finding was confirmed by Western analysis; flk-1/LacZ mixed-cell cultures treated with mEMAP II expressed 60% less ß-galactosidase (P = 0.06) (Figure 2E) than untreated controls.



View larger version (83K):
[in this window]
[in a new window]
  		Figure 2. A vascular network is formed in co-culture in the mesenchymal cells in regions of concentrated epithelial cyst formation. mEMAP II inhibits formation of the vascular network and induces the remaining endothelial cells to undergo a phenotypic change consistent with apoptosis. Co-cultures from fetal lungs containing the flk-1 LacZ transgene revealed that there is formation of a vascular network (using X-gal to reveal flk-1 LacZ cells and counterstained with fast red) in the mesenchymal cells in regions that have a high concentration of epithelial cyst formation (A, B) in vehicle-treated controls. The vascular network is not in the region that has been identified as laminin positive (arrows in B). Excess mEMAP II inhibited the formation of the vascular network (C, D) and induced the remaining endothelial cells to undergo nuclear condensation consistent with apoptosis (arrows in D) (n = 3/group on three different occasions). Our histologic observations were confirmed using Western analysis where mEMAP II decreased ß galactosidase activity by 60% as compared with control (E). Bar = 250 µm in A and C; 500 µm in B and D.

 
Because we have previously determined that mEMAP II induces apoptosis in endothelial cells, we sought to confirm cell death in mixed cell–type cultures exposed to mEMAP II. Cultures treated with mEMAP II as previously described were evaluated for micrographic evidence of apoptosis. After 72 h in culture, the remaining flk-1–expressing cells in treated cultures exhibited condensed nuclei characteristic of apoptosis (arrow, Figures 3C and 3D). This finding was confirmed by TUNEL assay. Cells were isolated from the fetal lungs of mice bearing the flk-1/LacZ transgene and maintained in mixed-cell culture for 72 h. Endothelial cells were identified by ß-galactosidase activity. A significantly greater proportion of the endothelial cells in mEMAP II treated cultures were apoptotic (Figures 3C and 3D) relative to untreated controls (arrows, Figures 3A and 3B).



View larger version (138K):
[in this window]
[in a new window]
  		Figure 3. mEMAP II induces apoptosis in the flk-1 LacZ-positive endothelial cells. We employed co-localization of x-gal using light microscopy and TUNEL assay using fluorescence to identify if the endothelial cells with the condensed nuclei in co-culture were indeed apoptotic. Consistent with our initial observations, we found that mEMAP II induced apoptosis in the flk-1 LacZ-positive endothelial cells identified by light microscopy (C) and TUNEL analysis (D). In contrast, no significant apoptosis was noted in the flk-1 LacZ cells in control culture (A, light microscopy; B, TUNEL analysis). Bar = 500 µm.

 
Neovascular Inhibition by mEMAP II Attenuates Epithelial Cyst Formation
The effects of mEMAP II on epithelial cyst formation were examined. Dissociated cells from E15.5 fetal lungs were observed for spontaneous recombination in a three-dimensional fashion into epithelial cyst for 72 h in the presence of mEMAP II, an adsorbing anti–EMAP II antibody, vehicle, or nonspecific rabbit IgG. A dose-dependent decrease in epithelial cyst formation was observed in cultures treated with exogenous mEMAP II (Figures 4C, 4D, and 4G), relative to unstimulated cultures (Figures 4A, 4B, and 4G) (slides were hematoxylin-stained and 10 high-power fields counted per slide and averaged per n with n = 3–5/group on three different occasions) (*P < 0.01). The few cysts that formed in the presence of excess mEMAP II were poorly organized (Figures 4C and 4D) relative to control (Figures 4A and 4B). Specifically, in addition to fewer cysts, those formed lacked the characteristic structured cyst formation, were rounded up, and were smaller in formation. Conversely, treatment of cultures with an adsorbing antibody against EMAP II was associated with increased epithelial cyst formation (Figures 4E, 4F, and 4H), relative to control (nonspecific rabbit IgG) (slides were hematoxylin-stained and 10 high-power fields counted per slide and averaged per n with n = 3–5/group on three different occasions). To confirm these results, cultures were subjected to keratin Western analysis. Keratin (18) is synthesized during epithelial differentiation, and expression correlates with epithelial cyst formation. Mixed cell–type cultures treated with mEMAP II had 20% less keratin expression, which trended toward statistical significance (P = 0.09) relative to unstimulated controls (data not shown). Thus, epithelial cyst formation in vitro is inversely related to mEMAP II concentration.




View larger version (168K):
[in this window]
[in a new window]
  		Figure 4. Vascular disruption inhibits the formation of epithelial cyst in co-culture. mEMAP II induced a dramatic dose dependent reduction in epithelial cyst formation (C, D, G) as compared with control (A, B, G) (n = 8/group performed on four different occasions). Furthermore, when one histologically (hematoxylin and eosin) compares the normal epithelial cyst formation (A, B) to that of mEMAP II treated (C, D), you can see that mEMAP II altered their ability to form concentric well formed cyst. Lastly, if you block the anti-vascular effects of mEMAP II by delivering a blocking antibody, you get the opposite response with a dose-dependent increase in cyst formation (E, F, H). *P < 0.01. Bar = 250 µm in A, C, and E; 500 µm in B, D, and F. (Continues on next page.)

 
Exogenous mEMAP II Does Not Modulate the Proliferation of Epithelial or Mesenchymal Cells
The anti-angiogenic peptide mEMAP II has been previously demonstrated to induce apoptosis in proliferating vascular endothelial cells (10, 11), suggesting its use as a neovascular inhibitor in this system. To evaluate its specificity, the effect of mEMAP II on the proliferation of fetal lung mesenchymal and epithelial cell isolates was evaluated. Cells were isolated at gestational ages empirically determined to yield the purest and most viable epithelial and mesenchymal cell populations. Mesenchymal cells were isolated from fetal lungs at 14–15 d after conception and epithelial cells from fetal lungs at 16–17 d postconceptual age. Cell purity was estimated by light microscopy observation of> 90% cell homogeneity and cytokeratin Western analysis. Cells were rested for 10 h after isolation, then exposed to mEMAP II 3.2µg/ml for up to 72 h. This concentration was previously determined to optimally induce the apoptosis of endothelial cells. Cell proliferation was evaluated by 3H-thymidine incorporation following 24, 48, and 72 h of mEMAP II. All groups were compared with the basal level of control 3H-thymidine incorporation and control incorporation was assigned the empiric number of 100%. mEMAP II had no significant effect on the proliferation of monocultured mesenchymal or epithelial cells, or on mixed co-culture cell populations in culture as compared with the basal proliferation present in the control population (Figure 5). Western analysis of cellular proliferation using an anti–proliferating cell nuclear antigen (PCNA) confirmed that mEMAP II had no significant effect on monocultured mesenchymal or epithelial cell division (data not shown). The specificity of mEMAP II suggests that its effects on epithelial cyst formation result from modulated angiogenesis and not from the direct stimulation of epithelial or mesenchymal populations.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Vascular inhibition has no effect on cellular proliferation. Monoculture populations of epithelial and mesenchymal cells and co-culture cells were exposed to either mEMAP II or vehicle and cellular proliferation evaluated. Basal incorporation in control groups was designated as 100%. Any increase or decrease in 3H thymidine incorporation was documented as percent of control above or below the basal 100%. We found that there was no difference in 3H thymidine incorporation between any of the cell populations and their respective controls at any of the time points evaluated (24, 48, and 72 h). This graph reflects the results at 48 h as percent of control.

 
Neovascular Inhibition by mEMAP II Is Followed by Apoptosis of Epithelial Cysts
We then sought to determine if the decreased epithelial cyst formation observed in mixed co-culture cell populations stimulated with mEMAP II was associated with increased apoptosis. Cultures were assessed morphologically and by TUNEL assay. Initially, co-cultures treated for 48 h with mEMAP II exhibited apoptosis in the cells adjacent to epithelial cyst structures (Figure 6D, arrows). As noted previously, cells expressing flk-1 localized to these regions (Figures 3C and 3D, arrows). Subsequently, the entire epithelial cyst became apoptotic at 72 h (Figures 6C and 6E, arrow). This was less common in unstimulated identical control cultures (Figures 6A and 6B). In cultures treated with mEMAP II (3.2 µg/ml), 90% of epithelial cysts exhibited apoptosis in > 50% of cells. In contrast, only 44% of the cysts in untreated controls showed the same degree of apoptosis (P < 0.016)(Figure 6F). In contrast, identical cultures treated with a blocking EMAP II antibody had a marked reduction in the proportion of apoptotic cells (Figure 6F). Presence of EMAP II blocking antibody significantly reduced the percent of apoptotic epithelial cysts to 14%, significantly less than the baseline apoptosis of control (P < 0.002) (Figure 6F).




View larger version (172K):
[in this window]
[in a new window]
  		Figure 6. Vascular inhibition induces apoptosis in co-culture. TUNEL assay indicated that mEMAP II induced an increased amount of apoptosis in a co-culture (C, D, E) as compared with control (A, B). The distribution of apoptosis was in both a circular pattern noted at 2 d (D) as well as clumped cells noted at Days 2 and 3 (E). The percent of epithelial cyst presence that were apoptotic was markedly increased in the presence of mEMAP II at 72 h (90%, P < 0.016, paired t test) as compared with control (44%) (F). Presence of the EMAP II blocking antibody, resulted in a significant reduction below baseline in the percent of epithelial cells that were apoptotic (P < 0.002) as compared with its control (F). Squares, vehicle; circles, EMAP II–treated; triangles, EMAP II antibody (10 high-powered fields counted per slide comparing light microscopy to fluorescence and averaged per n with n = 3–5/group on three different occasions). Bar = 250 µm in A and C; 500 µm in B, D, and E.

 
Exogenous mEMAP II Modulates Fibronectin Expression in Co-Culture
The effects of mEMAP II on extracellular matrix were examined. Dissociated cells from E15.5 fetal lungs were observed for spontaneous recombination in a three-dimensional fashion into epithelial cyst for 72 h in the presence of mEMAP II (3.2 µg/ml) or vehicle. Western analysis of protein lysates indicated that exogenous mEMAP II induced a 2.5-fold increase in fibronectin expression (P = 0.009, analyzed by densitometry) (Figure 7). Conversely, mEMAP II had no effect on laminin protein expression (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 7. Exogenous mEMAP II increases fibronectin protein expressions by 2.5-fold. Western analysis of combined co-culture cell populations indicated that treatment with exogenous mEMAP II increased fibronectin protein expression 2.5-fold (P = 0.009) as compared with vehicle (n = 5/group performed on two different occasions).

 
EMAP II Expression Is Regulated by Epithelial–Mesenchymal Contact
To determine if and where EMAP II was expressed in mixed cell-type cultures, we examined EMAP II mRNA and protein expression. Although EMAP II was detectable in all cell populations by in situ hybridization, EMAP II transcripts predominantly localized to the contact point of the epithelial cells and mesenchymal cells (Figures 8A and 8B, arrows). This localization was confirmed by immunohistochemistry. EMAP II protein expression was maximal at the points where epithelial cells contacted mesenchymal cells (indicated by arrows, Figures 8C and 8D), and was inversely correlated to the distance between mesenchymal and epithelial cell populations. Western analysis of mesenchymal cell monocultures, epithelial cell monocultures, and co-culture confirmed that the expression of the precursor form of EMAP II is induced by epithelial and mesenchymal cell interactions. Co-cultures expressed 3-fold more precursor EMAP II protein than isolated mesenchymal or epithelial cell populations (P <= 0.005, analyzed by densitometry) (Figures 8E and 8F).




View larger version (85K):
[in this window]
[in a new window]
  		Figure 8. EMAP II expression suggests that vascular mediators are regulated through epithelial–mesenchymal interactions. Using in situ hybridization (A, B) and immunohistochemistry (C, D), we determined that precursor EMAP II expression was increased as the proximity of the mesenchymal and epithelial cells narrowed with its highest expression being at the junction of the two cell populations. Western analysis (E, F) of isolated versus combined cell populations noted that precursor EMAP II expression was increased 3-fold in cell populations cultured together (E, lane Co) (P < 0.001; n = 3/group performed on three separate occasions) versus either mesenchymal (E, lane M) or epithelial (E, lane E) cells alone. Bar = 250 µm in A and C; 500 µm in B and D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although partitioning lung morphogenesis and neovascularization into individual processes is attractive, it is becoming more apparent that it is the interaction of these two components that contributes to normal lung formation. What is less clear, is the dependence of these two processes on each other. The use of recombined co-cultures offers a distinct advantage in addressing this issue by providing an environment that contains the primary cellular components, the cellular matrix, independence from other organs and the ability to manipulate the environment. These data suggest that endothelial cell structures develop in culture despite the absence of hemodynamic circulation, and that the formation of endothelial structures is associated with interactions between epithelial and mesenchymal cells. Moreover, the organization of epithelial and mesenchymal cells into cystic structures is disrupted when neovascular development is attenuated by the anti-angiogenic peptide mEMAP II. Conversely, epithelial cyst formation is accelerated by an EMAP II–blocking antibody. Although mEMAP II induces apoptosis of dividing endothelial cells (10, 11), it has minimal effects on the proliferation of monocultured epithelial or mesenchymal cell populations. In recombined co-cultures, however, stimulation with mEMAP II induces a sequential apoptosis of both the endothelial and epithelial components of cystic structures, suggesting that vascular structures are necessary to the maintenance of the cystic structure. Finally, pro form of EMAP II is transcriptionally upregulated at sites of epithelial–mesenchymal cell interaction.

Evidence Supporting Coordination between Lung Morphogenesis and Neovascularization
Synergism between neovascularization and lung morphogenesis is supported by previous reports in which inhibition of lung neovascularization by several methods results in altered lung morphogenesis in vivo (6–9, 23). In these reports, alveolarization was disturbed by an interruption in alveolar structural integrity, epithelial differentiation, and induced distal lung dysplasia. However, the mechanisms by which vascular cells regulate lung morphogenesis have been difficult to evaluate in these models. The use of epithelial cyst formation in vitro as a model for airspace formation in vivo permits us to segregate the effects of vascular cell signaling from those of altered vascular transport.

Although mesenchymal and epithelial cellular components are readily identifiable in co-culture, little is known regarding vascular formation in this system. Flk-1/LacZ trangenic mice provide a unique opportunity to identify the presence and progression of vascular networks. Similar to in vivo reports using flk-1/LacZ mice (24), we found a time-dependent progressive formation of a reticular vascular networks associated with epithelial cysts. The vascular networks developed among the mesenchymal cells surrounding the epithelial cysts and were predominantly one cell wide. Progression of vascular network formation, localization to regions of epithelial mesenchymal proximity, and in vitro similarity to in vivo location suggests that vascular network in co-culture simulates in vivo vascular formation.

To determine epithelial and vascular inter-dependence, we induced selective endothelial cell apoptosis in co-culture and examined epithelial cyst formation as a proxy of alveolar architectural development. mEMAP II was used in these assays because it has been shown to possess specific anti-angiogenic properties that targets only the subpopulation of migrating and dividing endothelial cells to undergo apoptosis (10, 11). Furthermore, mEMAP II had little effect on the proliferation of epithelial and mesenchymal monocultures. Using the epithelial cyst formation in vitro as a model for airspace formation in vivo permitted us to segregate the effects of vascular cell signaling from those of altered vascular transport. In this system, neovascular disruption by EMAP II prevents epithelial cyst formation and destabilizes preexisting epithelial cysts. The association of vascular cell regulation with modulated architectural development in vitro suggests that vascular cells directly regulate the development and maintenance of lung architecture.

Although the interdependence between endothelial and epithelial cells is poorly understood, the endothelial lead sequential induction of apoptosis in co-culture suggests a facilitation between the two entitites. This vascular–epithelial interdependence is supported by studies showing that pulmonary vascular inhibition (7, 23, 25) or overabundance (7–9, 26) results in epithelial disruption. To determine the etiology of this endothelial and epithelial interaction, we examined factors common to neovascularization and epithelial morphogenesis. The formation of the extracellular matrix by surrounding cellular components has been well documented as structurally contributing to epithelial and vascular development. In particular, laminin and fibronectin have both been shown to contribute to epithelial morphogenesis (27) and neovascularization (28). Although laminins play a role in epithelial proliferation, lung bud cleft/branching (27), epithelial cyst formation (19, 20, 29), and microvascular construction (30–32), vascular inhibition of co-culture recombinants had no change in laminin expression. Conversely, fibronectin expression was elevated in co-cultures treated with mEMAP II. Fibronectin has been shown to bind epithelial and endothelial cells (33) and is responsible for attachment, adhesion, migration, and cytodifferentiation (28, 34). Furthermore, the incorporation of fibronectin into the extracellular matrix has been shown to contribute to epithelial cell differentiation (34, 35). Microvessel elongation in response to fibronectin is a result of an adhesion-dependent migratory recruitment of endothelial cell (36). Increased fibronectin protein expression suggests a disruption in an extracellular matrix component. This is an area of our ongoing research.

Equally interesting is the relationship between precursor proteins and their cleavage products. Well-established anti-angiogenic proteins activated by their cleavage from larger secreted proteins and expressed during lung development are endostatin and mEMAP II. Endostatin is cleaved from the larger secreted extracellular matrix protein Collagen XVIII. Whereas endostatin inhibits endothelial cell proliferation and migration and induces apoptosis (37), its precursor collagen XVIII has been shown to positively regulate the extracellular matrix–dependent motility and morphogenesis of endothelial cells (37). We and others have shown that once mEMAP II is cleaved from its precursor, whose function to data is unclear, it has selective extracellular anti-angiogenic effects on the endothelium of the tumor vasculature (10, 11, 38). Although the process of mEMAP II cleavage is poorly understood, the high expression levels of the precursor EMAP II suggest that its precursor form may have an alternative function. It is possible that EMAP II modulates communications between epithelial and mesenchymal cells involved in epithelial cyst formation (14, 17, 19, 20, 39). For example, EMAP II signaling may modulate soluble factor expression or regulate target cell sensitivity to those factors. As noted previously, we believe that this possibility is less likely because EMAP II has no detectable effect on the proliferation or apoptosis of either epithelial or mesenchymal cells. Unfortunately, definitive consideration of this alternative awaits elucidation of the receptors, intracellular signaling pathways, secretion, and cleavage associated with EMAP II.

Taken together, these findings suggest that vascular structures represent a distinct cellular population that arises from the interaction of epithelial and mesenchymal cells, exerts critical regulation on the formation of cystic structures, and is subject to feedback inhibition by the epithelial–mesenchymal interface of the fully organized epithelial cyst. Although the focus of this work has been on EMAP II in developing lungs, we anticipate that additional angiogenic regulators will be found to regulate lung morphogenesis, and that vascular signals will be found to modulate the development of other branched organs.


   Acknowledgments
 
The authors thank Fongrang Zhang for her technical skills. Research supported in part by a Grant in Aid from the American Lung Association RG-084-N (M.A.S.), CI-001N (M.A.S.), the Webb-Berger Foundation (M.A.S.), NIH HL-60061 (M.A.S.), NIH HL-03981 (M.A.S.), and American Heart Association–GIA (M.K.L.).

Received in original form April 24, 2003

Received in final form November 7, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wessells, N. 1977. Tissue Interactions and Development. Benjamin, Meno Park, CA.
  2. Masters, J. R. 1976. Epithelial-mesenchymal interaction during lung development: the effect of mesenchymal mass. Dev. Biol. 51:98–108.[CrossRef][Medline]
  3. Shannon, J. M., L. D. Nielsen, S. A. Gebb, and S. H. Randell. 1998. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev. Dyn. 212:482–494.[CrossRef][Medline]
  4. Carmeliet, P. 2000. Developmental biology: one cell, two fates [news]. Nature 408:43–45.[CrossRef][Medline]
  5. Yamashita, J., H. Itoh, M. Hirashima, M. Ogawa, S. Nishikawa, T. Yurugi, M. Naito, and K. Nakao. 2000. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408:92–96.[CrossRef][Medline]
  6. Jakkula, M., T. Cras, and S. Abman. 1999. Effects of fumagillin and talidomide treatment on alveolarization in the developing rat lung. Pediatr. Res. 45:67A. (Abstr.)
  7. Schwarz, M. A., F. Zhang, S. Gebb, V. Starnes, and D. Warburton. 2000. EMAP II inhibits lung neovascularization and airway epithelial morphogenesis. Mech. Dev. 95:123–132.[CrossRef][Medline]
  8. Zeng, X., S. E. Wert, R. Federici, K. G. Peters, and J. A. Whitsett. 1998. VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. Dev. Dyn. 211:215–227.[CrossRef][Medline]
  9. Zhou, L., C. R. Dey, S. E. Wert, and J. A. Whitsett. 1996. Arrested lung morphogenesis in transgenic mice bearing an SP-C–TGF-beta 1 chimeric gene. Dev. Biol. 175:227–238.[CrossRef][Medline]
  10. Berger, A. C., H. R. Alexander, G. Tang, P. S. Wu, S. M. Hewitt, E. Turner, E. Kruger, W. D. Figg, A. Grove, E. Kohn, D. Stern, and S. K. Libutti. 2000. Endothelial monocyte activating polypeptide II induces endothelial cell apoptosis and may inhibit tumor angiogenesis. Microvasc. Res. 60:70–80.[CrossRef][Medline]
  11. Schwarz, M. A., J. Kandel, G. Brett, J. Li, J. Hayward, R. E. Schwarz, O. Chappey, J. Wautier, J. Chabot, P. Lo Gerfo, and D. Stern. 1999. Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth, and induces apoptosis in growing endothelial cells. J. Exp. Med. 190:341–353.[Abstract/Free Full Text]
  12. Schwarz, M., M. Lee, F. Zhang, J. Zhao, Y. Jin, S. Smith, J. Bhuva, D. Stern, D. Warburton, and V. Starnes. 1999. EMAP II: a modulator of neovascularization in the developing lung. Am. J. Physiol. 276:L365–L375.
  13. Bissell, M. J., V. M. Weaver, S. A. Lelievre, F. Wang, O. W. Petersen, and K. L. Schmeichel. 1999. Tissue structure, nuclear organization, and gene expression in normal and malignant breast. Cancer Res. 59:1757s–1763s; discussion 1763s-1764s.[Abstract/Free Full Text]
  14. Schuger, L., J. Varani, P. D. Killen, A. P. Skubitz, and K. Gilbride. 1992. Laminin expression in the mouse lung increases with development and stimulates spontaneous organotypic rearrangement of mixed lung cells. Dev. Dyn. 195:43–54.[Medline]
  15. Post, M., and B. T. Smith. 1988. Histochemical and immunocytochemical identification of alveolar type II epithelial cells isolated from fetal rat lung. Am. Rev. Respir. Dis. 137:525–530.[Medline]
  16. Post, M., J. S. Torday, and B. T. Smith. 1984. Alveolar type II cells isolated from fetal rat lung organotypic cultures synthesize and secrete surfactant-associated phospholipids and respond to fibroblast-pneumonocyte factor. Exp. Lung Res. 7:53–65.[Medline]
  17. Schuger, L., J. Varani, R. Mitra, Jr., and K. Gilbride. 1993. Retinoic acid stimulates mouse lung development by a mechanism involving epithelial-mesenchymal interaction and regulation of epidermal growth factor receptors. Dev. Biol. 159:462–473.[CrossRef][Medline]
  18. Schuger, L., K. S. O'Shea, B. B. Nelson, and J. Varani. 1990. Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: involvement of laminin. Development 110:1091–1099.[Abstract/Free Full Text]
  19. Schuger, L., A. P. Skubitz, J. Zhang, L. Sorokin, and L. He. 1997. Laminin alpha1 chain synthesis in the mouse developing lung: requirement for epithelial-mesenchymal contact and possible role in bronchial smooth muscle development. J. Cell Biol. 139:553–562.[Abstract/Free Full Text]
  20. Schuger, L., P. Yurchenco, N. K. Relan, and Y. Yang. 1998. Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization. Int. J. Dev. Biol. 42:217–220.[Medline]
  21. Zhang, F., and M. A. Schwarz. 2000. Temporo-spatial distribution of endothelial-monocyte activating polypeptide II, an anti-angiogenic protein, in the mouse embryo. Dev. Dyn. 218:490–498.[CrossRef][Medline]
  22. Schuger, L., S. O'Shea, J. Rheinheimer, and J. Varani. 1990. Laminin in lung development: effects of anti-laminin antibody in murine lung morphogenesis. Dev. Biol. 137:26–32.[CrossRef][Medline]
  23. Jakkula, M., T. D. Le Cras, S. Gebb, K. P. Hirth, R. M. Tuder, N. F. Voelkel, and S. H. Abman. 2000. Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L600–L607.[Abstract/Free Full Text]
  24. Schachtner, S. K., Y. Wang, and H. Scott Baldwin. 2000. Qualitative and quantitative analysis of embryonic pulmonary vessel formation. Am. J. Respir. Cell Mol. Biol. 22:157–165.[Abstract/Free Full Text]
  25. Lin, Y., S. Zhang, M. Rehn, P. Itaranta, J. Tuukkanen, R. Heljasvaara, H. Peltoketo, T. Pihlajaniemi, and S. Vainio. 2001. Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C. Development 128:1573–1585.[Abstract]
  26. Brown, K. R., K. M. England, K. L. Goss, J. M. Snyder, and M. J. Acarregui. 2001. VEGF induces airway epithelial cell proliferation in human fetal lung in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L1001–L1010.[Abstract/Free Full Text]
  27. Mollard, R., and M. Dziadek. 1998. A correlation between epithelial proliferation rates, basement membrane component localization patterns, and morphogenetic potential in the embryonic mouse lung. Am. J. Respir. Cell Mol. Biol. 19:71–82.[Abstract/Free Full Text]
  28. Risau, W., and V. Lemmon. 1988. Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev. Biol. 125:441–450.[CrossRef][Medline]
  29. Schuger, L. 1997. Laminins in lung development. Exp. Lung Res. 23:119–129.[Medline]
  30. Gonzalez, A. M., M. Gonzales, G. S. Herron, U. Nagavarapu, S. B. Hopkinson, D. Tsuruta, and J. C. Jones. 2002. Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo. Proc. Natl. Acad. Sci. USA 99:16075–16080.[Abstract/Free Full Text]
  31. Sanz, L., L. Garcia-Bermejo, F. J. Blanco, P. Kristensen, M. Feijoo, E. Suarez, B. Blanco, and L. Alvarez-Vallina. 2003. A novel cell binding site in the coiled-coil domain of laminin involved in capillary morphogenesis. EMBO J. 22:1508–1517.[CrossRef][Medline]
  32. Thyboll, J., J. Kortesmaa, R. Cao, R. Soininen, L. Wang, A. Iivanainen, L. Sorokin, M. Risling, Y. Cao, and K. Tryggvason. 2002. Deletion of the laminin alpha4 chain leads to impaired microvessel maturation. Mol. Cell. Biol. 22:1194–1202.[Abstract/Free Full Text]
  33. Caniggia, I., J. Liu, R. Han, J. Wang, A. K. Tanswell, G. Laurie, and M. Post. 1996. Identification of receptors binding fibronectin and laminin on fetal rat lung cells. Am. J. Physiol. 270:L459–L468.
  34. Swisher, J. W., and D. E. Rannels. 1997. Assembly of exogenous fibronectin into type II cell extracellular matrix. Am. J. Physiol. 272:L908–L915.
  35. Arai, H., H. Hirano, S. Mushiake, M. Nakayama, G. Takada, and K. Sekiguchi. 1997. Loss of EDB+ fibronectin isoform is associated with differentiation of alveolar epithelial cells in human fetal lung. Am. J. Pathol. 151:403–412.[Abstract]
  36. Nicosia, R. F., E. Bonanno, and M. Smith. 1993. Fibronectin promotes the elongation of microvessels during angiogenesis in vitro. J. Cell. Physiol. 154:654–661.[CrossRef][Medline]
  37. Kuo, C. J., K. R. LaMontagne, Jr., G. Garcia-Cardena, B. D. Ackley, D. Kalman, S. Park, R. Christofferson, J. Kamihara, Y. H. Ding, K. M. Lo, S. Gillies, J. Folkman, R. C. Mulligan, and K. Javaherian. 2001. Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain. J. Cell Biol. 152:1233–1246.[Abstract/Free Full Text]
  38. Ferrario, A., K. Tiehl, N. Rucker, M. Schwarz, P. Gill, and C. Gomer. 2000. Anti-angiogenic treatment enhances photodynamic therapy responsiveness in a mouse mammary carcinoma. Cancer Res. 60:4066–4069.[Abstract/Free Full Text]
  39. Schuger, L., A. P. Skubitz, K. S. O'Shea, J. F. Chang, and J. Varani. 1991. Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development. Dev. Biol. 146:531–541.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J.-R. Tang, G. Seedorf, V. Balasubramaniam, A. Maxey, N. Markham, and S. H. Abman
Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1271 - L1280.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. J. Mondrinos, S. Koutzaki, P. I. Lelkes, and C. M. Finck
A tissue-engineered model of fetal distal lung tissue
Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L639 - L650.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0145OCv1
30/6/784    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwarz, M. A.
Right arrow Articles by Lee, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwarz, M. A.
Right arrow Articles by Lee, M. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.