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Am J Physiol Lung Cell Mol Physiol 288: L167-L178, 2005. First published September 17, 2004; doi:10.1152/ajplung.00185.2004
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Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung

Minke van Tuyl,1,2 Jason Liu,1 Jinxia Wang,1 Maciek Kuliszewski,1 Dick Tibboel,2 and Martin Post1

1Lung Biology Program, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario, Canada; and 2Department of Pediatric Surgery, Sophia Children's Hospital, Erasmus Medical Center, Rotterdam, The Netherlands

Submitted 19 May 2004 ; accepted in final form 14 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1{alpha} or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

angiogenesis; vasculogenesis; airway branching; lung development

THE MOLECULAR BASE of pulmonary development has been the subject of extensive research over the past few decades. Many growth and transcription factors and morphogens have been shown to play key roles in pulmonary development, regulating trachea and lobe formation, branching morphogenesis, proximo-distal epithelial differentiation, and mesenchymal development (64). Pulmonary vascular development has gained substantial interest over the past few years because of the putative vascular involvement in neonatal diseases such as respiratory distress syndrome and bronchopulmonary dysplasia (1, 14, 54). Furthermore, it has been postulated that lung abnormalities in congenital diaphragmatic hernia have a vascular origin (13, 65). Two processes have been implicated in vascular development: angiogenesis, which is the sprouting of new vessels from preexisting ones, and vasculogenesis, which is the development of blood lakes de novo that transform into vessels (57). Both processes are believed to occur during pulmonary vascular development (31). Recently, it has been shown that vascular development takes place during all stages of lung development (9).

It is important to realize that normal pulmonary development takes place in a relative hypoxic environment of the uterus (42). Several studies have shown that the low-fetal oxygen environment is beneficial for embryo development (12, 46) and for cardiovascular (72) and kidney (44) organogenesis, but relatively little is known about the influence of oxygen on fetal lung development. Preliminary studies in rats have shown that a fetal oxygen tension (hypoxia) maintains lung morphogenesis in vitro (24). Midtrimester human fetal lung explants cultured at fetal oxygen tension had increased expression of vascular endothelial growth factor (VEGF) A compared with explants cultured at ambient oxygen cultures (2). VEGF is a potent mitogen for endothelial cells, influencing angiogenesis and vasculogenesis (52). VEGF expression is regulated by hypoxia-inducible factor (HIF)-1{alpha}, which encodes a transcription factor that is expressed in most, if not all, cells in response to hypoxia (48, 61). Moreover, HIF-1{alpha} is essential for embryonic vascularization and survival, hypoxia-induced pulmonary vascular remodeling, and tumor vascularization (36). In Drosophila, oxygen is delivered to the cells via an extensive network of tubules that deliver oxygen directly to the cells without interference of a vascular system (27). The master gene in regulating tracheal cell specification in Drosophila is the basic-helix-loop-helix (bHLH)-PAS protein Trachealess, which displays high homology to mammalian bHLH-PAS proteins, including HIF-1{alpha} (32, 71). Interestingly, a basic amino acid sequence immediately before the NH2 terminus of the HLH domain, which is known to be the site for DNA recognition, is completely conserved between HIF-1{alpha} and Trachealess (32, 71). Thus a protein involved in the hypoxic response and vascular development in mammalians may regulate tracheal development in Drosophila. Recent studies have demonstrated that fetal oxygen tension indeed stimulates the branching of the Drosophila tracheal system (35).

Because a hypoxic environment is critical for vascularization, it is feasible that lung airway branching morphogenesis in utero is controlled by oxygen-regulated pulmonary vascular development. Recent reports have indeed suggested an active role for vascularization in lung development. Schwarz et al. (59) showed that inhibition of neovascularization with endothelial monocyte activating polypeptide II (EMAPII) resulted in an arrest of lung airway epithelial morphogenesis. Furthermore, overexpression of the VEGF isoform 164 in distal airway epithelium of the developing lung resulted in an increased (73) or decreased (4) peritubular vascularity, depending on the time of VEGF 164 overexpression. In both studies, gross abnormalities in lung branching morphogenesis were noted, with a concomitant decrease in epithelial acinar tubules and mesenchyme (4, 73).

The objectives of the present study were to investigate the influence of fetal physiological oxygen tension on the developing lung and to determine the interaction between the developing vascular bed and the pulmonary epithelium.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Transgenic mice. Tie2-LacZ mice were obtained from Jackson Laboratory (Bar Harbor, ME) (58). In Tie2-Lac transgenic mice, the 2.1-kb 5'-flanking region of the murine Tie2 promoter drives the expression of the bacterial LacZ reporter gene exclusively to endothelial cells (58). In C101-LacZ or cordon blue-LacZ transgenic mice, the C101 promoter drives the LacZ gene expression specifically to epithelial cells (22). Cells transcribing the LacZ gene can be viewed by staining for {beta}-galactosidase activity. Both transgenic lines were maintained on a CD1 background. All mouse protocols were in accordance with Canadian Counsel of Animal Care guidelines and were approved by the Animal Care and Use Committee of the Hospital for Sick Children, Toronto, Ontario, Canada.

Whole lung organ culture. Lung buds were dissected from embryonic day (E) 11.5 CD1, C101-LacZ, or Tie2-LacZ mouse embryos (day of vaginal plug is E0.5) and placed on a floating (8-µm Whatman Nuclepore polycarbonate) membrane (Integra Environmental, Burlington, ON, Canada). Explants were grown in DMEM supplemented with 10% FCS (GIBCO, Grand Island, NY) and maintained in an atmosphere of either 3% O2/92% N2/5% CO2 or 20% O2/75% N2/5% CO2 at 37°C. Antisense oligonucleotides (ODNs) targeted against the translation initiation site of murine HIF-1{alpha} (antisense: 5'-TGCCGTCGCCGCCATC-3', sense: 5'-GATGGCGGCGACGGCA-3') and VEGF (16) were added to the medium in a final concentration of 20 µM. In VEGF rescue experiments, recombinant VEGF (R&D Systems) was added to lung explants in a final concentration of 100 ng/ml. The medium, ODNs, and VEGF were changed every other day. In control lung explants, lung growth and branching morphogenesis proceeded as described previously (66).

X-galactosidase staining. Cultured LacZ-lung explants were fixed (1% formaldehyde, 0.1% glutaraldehyde, 2 mM MgCl2, and 5 mM EGTA in 0.1 M sodium phosphate buffer, pH 7.8–8.0, for 45 min at 4°C), washed (2 mM MgCl2, 0.01% deoxycholate, and 0.02% Nonidet P-40 in 0.1 M sodium phosphate buffer, pH 7.8–8.0) 4x 30 min at 4°C, and stained overnight at 37°C in X-galactosidase (X-gal) staining solution [5 mM K4Fe(CN)6:3H2O and 5 mM K3Fe(CN)6 in wash buffer, mixed 40:1 with X-gal stock solution (40 mg/ml in dimethyl formamide)]. Explants were washed in 70% ethanol, fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C, and stored in 70% ethanol. For imaging, explants were dehydrated 2x 30 min in methanol and cleared in methyl salicylate. For sectioning, explants were dehydrated in a graded series of ethanol, kept overnight in 1-butanol, embedded in Paraplast, and mounted onto Superfrost slides (Fisher Scientific, Unionville, ON, Canada). Pictures were taken with a Leica digital imaging system.

Immunostaining for platelet endothelial cell adhesion molecule. Cultured CD1 lung explants were fixed overnight in 4% PFA in PBS at 4°C. Explants were washed twice in PBS, dehydrated through a graded series of ethanol, kept overnight in 1-butanol, and embedded in Paraplast. Seven-micrometer-thick sections were cut, and endogenous peroxidase activity was quenched with 0.15% hydrogen peroxide in methanol. Sections were incubated with trypsin (0.6 mg/ml) for 5 min at room temperature (RT). Nonspecific binding sites were blocked using 5% normal goat serum and 1% bovine serum albumin followed by overnight incubation at 4°C with rat anti-mouse CD31 antibody [1:60; platelet endothelial cell adhesion molecule (PECAM)-1; BD Biosciences Pharmingen, Mississauga, ON, Canada] and incubation with biotinylated secondary sheep anti-rat antibody (1:300) at RT. Color detection was performed according to the instructions in the Vectastain ABC and DAB kit (Vector Laboratories, Burlingame, CA). Slides were lightly counterstained with Carazzi hematoxylin. Pictures were taken using a Leica digital imaging system.

Whole mount in situ hybridization. Whole mount in situ hybridization was performed essentially as described by Riddle et al. (56). Briefly, cultured CD1 lung explants were fixed overnight in 4% PFA in PBS at 4°C. Explants were washed in PBS-T (PBS containing 0.1% Tween 20), dehydrated in graded series of methanol in PBS-T, and stored in 100% methanol. After rehydration, explants were bleached in hydrogen peroxide, treated with proteinase K (20 µg/ml), postfixed in 4% PFA and 0.2% glutaraldehyde, and prehybridized for 1 h at 70°C. Explants were then hybridized with the appropriate digoxigenin (DIG)-labeled riboprobe (1 µg/ml) overnight at 70°C. After washes in 50% formamide, 5x SSC, pH 4.5, and 1% SDS at 70°C followed by washes in 50% formamide, 2x SSC, pH 4.5, at 65°C, and Tris-buffered saline (TBS)-T (TBS containing 1% Tween 20) at RT, explants were preblocked with sheep serum in TBS-T and subsequently incubated with anti-DIG alkaline phosphatase 1:5,000 in blocking solution (Roche, Montreal, Quebec, Canada) at 4°C. The next day, explants were washed in PBS-T followed by washes in NTM-T (100 mM NaCl, 100 mM Tris, pH 9.5, 50 mM MgCl2, and 0.1% Tween) and then incubated with nitro blue tetrazolium chromogen/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche) at RT until purple color appeared. After color development, explants were washed in NTM-T and PBS-T, dehydrated in a graded series of methanol in PBS-T, and stored in PBS-T at 4°C. Pictures were taken using a Leica digital imaging system.

Tissue section in situ hybridization. Cultured CD1 lung explants were fixed overnight in 4% PFA in PBS, dehydrated in ethanol, and embedded in Paraplast. Sections of 12 µm were cut and mounted on Superfrost slides (Fisher Scientific). Explants were then assayed for nonradioactive RNA in situ hybridization according to Moorman et al. (50). Briefly, after dewaxing and rehydrating, tissue sections were permeabilized with proteinase K (20 µg/ml), postfixed in 4% PFA and 0.2% glutaraldehyde, and prehybridized for 1 h at 70°C. The sections were hybridized overnight at 70°C with a DIG-labeled riboprobe for surfactant protein C (1 µg/ml). The next day, sections were washed in 50% formamide in 2x SSC, pH 4.5, at 65°C followed by PBS-T washes. Subsequently, the sections were incubated with anti-DIG alkaline phosphatase 1:1,000 in blocking solution (Roche) at 4°C. The next day, sections were washed in PBS-T followed by washes in NTM and then incubated with NBT/BCIP (Roche) at RT until the purple color appeared (4–5 h). All slides were stopped at the same time to make comparison over different stages of development possible. After color development, sections were washed in distilled water, dehydrated in a graded series of ethanol and xylene, and mounted with coverslips using permount (Fisher Scientific). Pictures were taken using a Leica digital imaging system.

RNA isolation and real-time RT-PCR. CD1 lung explants, cultured at 3 or 20% oxygen, were rinsed in ice-cold PBS after 2, 4, and 6 days in culture (12 lungs from 3 litters at 2 and 4 days and 9 lungs from 2 litters at 6 days), immediately frozen in liquid nitrogen, and stored at –70°C. Explants from each time group were divided into three groups, and RNA was extracted using the RNA easy kit (Qiagen, Mississauga, ON). One microgram of RNA was reverse transcribed (37°C) using random hexamers (Applied Biosystems, Foster City, CA). The resulting templates (50 ng of cDNA for target genes and 5 ng for 18S) were quantified by real-time PCR (ABI Prism 7700). Primers and TaqMan probes for total VEGF, VEGF receptor Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1 or Vegfr2), angiopoietin (Ang)-1, and Ang-2 were similar to previously published sequences (17), whereas primers and TaqMan probes for PECAM-1, Tie2, fibroblast growth factor-10 (FGF-10) and FGF receptor 2 (Fgfr2) (for both Fgfr2-IIIb and -IIIc isoforms) were purchased from ABI as Assays-on-Demand for murine genes. For each probe, a dilution series determined the efficiency of amplification of each primer-probe set, and the relative quantification method was employed (43). For the relative quantitation, PCR signals were compared among groups after normalization using 18S as an internal reference. Briefly, relative expression was calculated as 2-(Ctgene of interest–Ct18S) and fold change was calculated according to Livak and Schmittgen (43). P < 0.05 was considered statistically significant.

Western blotting. Fifty micrograms of total protein were subjected to 10% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. Nonspecific binding was blocked by incubation in 5% (wt/vol) nonfat dry milk in TBS containing 0.1% (vol/vol) Tween 20 (TBS-T) for 60 min. Membranes were then incubated with mouse monoclonal anti-HIF-1{alpha} (mgc3, 1:100; Affinity Bioreagents, Golden, CO). After overnight incubation, membranes were washed with TBS-T and incubated for 60 min at RT with 1:10,000 rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 5% (wt/vol) nonfat dry milk in TBS-T. After being washed with TBS-T, blots were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Low oxygen enhances epithelial branching morphogenesis. The effect of low oxygen on epithelial branching in vitro was investigated using C101-LacZ mice. Lungs were dissected at E11.5 and cultured at either 3 or 20% oxygen. Whole mount LacZ staining revealed a complete image of the developing airways. On E11.5, the lung rudiments consisted of two epithelial buds that, over time (48 h) in culture, progressed toward two main airways (Fig. 1A) and four lobes on the right and one lobe on the left side of the trachea. Additional branching (96 h) provided the exponential growth and complexity of distal airways (Fig. 1, C and D). LacZ staining of lung explants cultured at 3% oxygen revealed a more elaborated distal branching (Fig. 1, B and D) when compared with explants cultured at 20% oxygen (Fig. 1, B and D vs. A and C, respectively). Because C101-LacZ is solely expressed in epithelial cells of the airways and not in mesenchymal or endothelial cells, these results suggest that a low-oxygen environment benefits epithelial branching morphogenesis.



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  		Fig. 1. Low oxygen enhances epithelial branching morphogenesis in vitro. C101-LacZ expression in embryonic day (E) 11.5 lung explants after 48 (A and B) and 96 (C and D) h in culture. Explants cultured at 3% oxygen (B and D) showed more complex branching compared with explants cultured at 20% oxygen (A and C). *Shows location of the trachea; arrowheads show the main bronchi, and the arrows show increased distal branch tips. Blue color is X-galactosidase (X-gal) staining in airway epithelial cells. Bars, 100 µm.

 
In Drosophila tracheal development, terminal branching is mainly controlled by oxygen. A low oxygen tension stimulates terminal branch formation, and high oxygen tension suppresses it (35). Oxygen-starved cells were shown to generate a signal that functions as a chemoattractant for terminal branches. This signal was identified as Branchless, the Drosophila ortholog for FGF (35). In mammalian development, FGF-10 and its receptor Fgfr2 play a role in airway branching reminiscent of that described for Branchless and its receptor Breathless in Drosophila trachea development (67). Both FGF-10 and Fgfr2 have been shown to be indispensable for murine lung development (49). Based on these data, we tested the possibility that increased airway branching morphogenesis in lung explants cultured at 3% oxygen was caused by an increase in FGF-10/Fgfr2 signaling. Whole mount in situ hybridization was performed to investigate FGF-10 mRNA expression in lung explants. FGF-10 was mainly expressed in the mesenchyme of distal airways of E11.5 lung explants cultured for 24 h at 20% oxygen (Fig. 2A). FGF-10 was unevenly expressed, i.e., high levels of expression were detected in the mesenchyme between the distal buds, and low expression was detected at the distal branch tips (Fig. 2, A and B). A similar spatial expression pattern has been reported for the developing murine lung in vivo (6). Explants cultured at 3% oxygen demonstrated a similar spatial expression pattern as those kept at 20% oxygen (Fig. 2, B vs. A). To confirm these results, we performed real-time RT-PCR for FGF-10 and Fgfr2 in explants cultured at either 3 or 20% oxygen for 2, 4, and 6 days. No significant changes in gene expression of both signaling molecules were found between explants cultured at 3 or 20% oxygen (Fig. 2, C and D).



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  		Fig. 2. Low oxygen does not affect fibroblast growth factor (FGF)-10 and bone morphogenetic protein-4 (Bmp4) expression. Whole mount in situ hybridization for FGF-10 (A and B) and Bmp4 (E and F) mRNA in lung explants cultured for 24 h at 20% (A and E) or 3% oxygen (B and F) is shown. FGF-10 was highly expressed in the mesenchyme between branches (arrows), but expressed less in distal branch tips (arrowheads). Bmp4 was strongly expressed in the epithelium of distal branch tips (arrows). Low oxygen did not affect the spatial expression of FGF-10 or Bmp4. Real-time RT-PCR (n = 3 from 3 separate experiments and expressed as fold change ± SD relative to explants cultured at 20% oxygen for 2 days) revealed no significant differences in the mRNA expression of FGF-10 (C) or its receptor Fgfr2 (D). Section in situ hybridization for surfactant protein C (SP-C) mRNA in explants cultured for 96 (G and H) or 144 h (I and J) at 20% (G and I) or 3% oxygen (H and J) revealed enhanced SP-C expression in the distal parts of explants cultured at 3% oxygen (H and J). Bars, 125 (A, B, E, and F) and 100 µm (G–J).

 
In addition to FGF-10, other growth factors, including bone morphogenetic protein-4 (Bmp4), have been shown to be required for the coordinated branching and elongation of airway tubules during lung development (69). Therefore, we performed whole mount in situ hybridization for Bmp4 to investigate whether culturing of lung explants at 3% oxygen influenced Bmp4 expression. In explants cultured for 48 h at 20% oxygen, Bmp4 was mainly expressed in the pulmonary epithelium with marked enhanced expression at the distal branch tips (Fig. 2E). This spatial expression pattern is similar to that of the embryonic murine lung in vivo (7). Bmp4 expression in explants cultured at 3% oxygen showed a similar spatial expression pattern (Fig. 2F). The increased number of Bmp4-positive distal branch tips in lung explants cultured at 3% oxygen reflects the enhanced airway branching of the lung in low oxygen (Fig. 2F). Together, the results suggest that the increased epithelial branching morphogenesis of explants cultured at 3% oxygen is likely not due to changes in FGF-10 and Bmp4 signaling.

Distal airway epithelium is normally lined with SP-C-positive cells. To investigate whether low oxygen maintained epithelial differentiation, we performed section in situ hybridization for SP-C on E11.5 explants cultured at either 3 or 20% oxygen. In lung explants cultured at 20% oxygen, SP-C mRNA was detected in the distal epithelium (Fig. 2, G and I, for 96 and 144 h, respectively). Explants cultured at 3% oxygen demonstrated enhanced SP-C expression in distal epithelial branches (Fig. 2, H and J, for 96 and 144 h, respectively). These results are in line with a preliminary report by Gebb and Shannon (23) and indicate that explants cultured at 3% oxygen maintain their appropriate epithelial morphogenesis.

Low oxygen enhances vascular development. The effect of hypoxia on the development of the pulmonary vascular system in vitro was investigated using Tie2-LacZ mice. Lungs were dissected at E11.5 and cultured at either 3 or 20% oxygen. Whole mount LacZ staining revealed a complete image of the developing vascular bed. Explants cultured for 48 h at 3% oxygen showed a more complex vascular network in the mesenchyme surrounding the developing lung buds than explants cultured at 20% oxygen (Fig. 3, B vs. A). The enhanced vascularization in 3% oxygen explants was even more evident after 72 h of culture (Fig. 3, D vs. C). Noteworthy is the arrangement of the vessels between the explants cultured at different oxygen tensions. In explants cultured at 20% oxygen, X-gal-positive vessels were detected along the trachea, main bronchi, and smaller distal airways. No vascularization, however, was detected along the distal branch tips (edges) of the explants (Fig. 3C). In explants cultured at 3% oxygen, however, X-gal-positive vessels were detected along the trachea, main bronchi, and smaller airways up to the distal branch tips (edges) of the explants (Fig. 3D). Sectioning of the explants confirmed the increased extent of the vascular bed in explants cultured at 3% oxygen (Fig. 3F) when compared with explants cultured at 20% oxygen (Fig. 3E). These results indicate that low oxygen enhances vascular development in embryonic lung explants and stimulates vascular growth in the periphery of the lung, thereby covering all distal airways.



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  		Fig. 3. Low oxygen stimulates pulmonary vascular development in vitro. Tie-LacZ expression in E11.5 lung explants after 48 (A and B) and 72 (C–F) h in culture. Explants cultured at 3% oxygen (B and D) showed more complex vascular branching compared with explants cultured at 20% oxygen (A and C). In explants kept at 3% oxygen, vessels extended from the trachea (arrows) to the most distal branch tips (arrowheads), whereas in explants kept at 20% oxygen, the vessels did not reach the distal branch tips (arrowheads). Sectioning of E11.5 Tie-LacZ lung explants after 48 h in culture confirmed enhanced X-gal staining in explants cultured at 3% oxygen (F) compared with explants cultured at 20% oxygen (E). Sections were counterstained with eosin. Blue color is X-gal staining in endothelial cells. Bars, 100 µm.

 
Low oxygen increases PECAM-1 and VEGF expression. To confirm the increase in vascularization in Tie2-LacZ lung explants cultured at 3% oxygen, we performed immunohistochemistry for PECAM-1 (CD31), an endothelial cell marker. After 72 h in culture, greater PECAM-1-immunopositive reactivity was observed in the mesenchyme from explants cultured at 3% oxygen (Fig. 4B) compared with explants cultured at 20% oxygen (Fig. 4A). Note the more complex pattern of tubular epithelial branching in explants cultured at 3% oxygen (Fig. 4B), consistent with the increased airway branching seen in C101-LacZ explants cultured at 3% oxygen (Fig. 1, B and D). We then performed real-time RT-PCR for PECAM-1 in E11.5 lung explants cultured for 2, 4, and 6 days at either 3 or 20% oxygen. Culturing explants at 3% oxygen significantly increased PECAM-1 mRNA expression compared with explants cultured at 20% oxygen (Fig. 4C). These results confirm that low oxygen stimulates vascular development in embryonic lung explants.



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  		Fig. 4. Low oxygen stimulates platelet endothelial cell adhesion molecule (PECAM)-1 and VEGF expression. PECAM-1 (CD31) immunohistochemistry is shown in E11.5 lung explants after 72 h in 3% (B) or 20% oxygen (A) culture. Sections were counterstained with Carazzi hematoxylin. Explants cultured at 3% oxygen (B) showed increased PECAM-1 staining compared with explants cultured at 20% oxygen (A). Brown color (arrows) is positive staining. Real-time RT-PCR (n = 3 from 3 different experiments and expressed as fold change ± SD relative to explants cultured at 20% oxygen for 2 days) showed increased expression of PECAM-1 (C) and VEGF (D) in explants cultured at 3% oxygen. Vegfr2 (Flk-1) expression (E) was initially upregulated in explants at low oxygen, but its expression was not different from 20% oxygen explants when cultured for longer periods. *P < 0.05. Bars, 250 µm.

 
Many genes that are involved in angiogenesis and vessel growth are upregulated by hypoxia, including VEGF (62). Because a hypoxic environment increased vascular density in cultured lung explants, we performed real-time RT-PCR for total VEGF in E11.5 lung explants. VEGF mRNA was dramatically upregulated in explants exposed to 3% oxygen compared with lung explants cultured at 20% oxygen (Fig. 4D). Expression of Vegfr2/Flk-1 was initially (2- and 4-day culture) upregulated in lung explants kept at 3% oxygen compared with explants cultured at 20% oxygen, but no differences in Vegfr2/Flk-1 transcript levels were noted between lung explants cultured for longer times (6 days; Fig. 4E). The expression levels of other angiogenic cytokines, such as Ang-1 and Ang-2, were not affected by low oxygen (not shown).

Vascular development influences epithelial branching morphogenesis. To investigate whether vascularization affects epithelial branching morphogenesis, we inhibited vascularization using antisense ODNs targeting either VEGF or HIF-1{alpha}. E11.5 lung explants from Tie2-LacZ mice were cultured at 3% oxygen in the presence or absence of either antisense ODNs. After 48 h in culture, intense vascular LacZ staining was detected in control and sense HIF-1{alpha} ODN-treated lung explants (Fig. 5, A and B, respectively). A fine vascular network wrapped around the trachea and main bronchi and covered the epithelial tubules as far as the distal branch tips. In contrast, antisense HIF-1{alpha} ODN-treated lung explants showed a dramatic decrease in vascular development (Fig. 5C). Although vessels ran from the trachea area down along the main bronchi and some proximal epithelial branches, most of the peripheral tissue was devoid of vessels (Fig. 5C). Antisense but not sense HIF-1{alpha} ODN completely abolished HIF-1{alpha} protein expression in the lung explants kept at 3% oxygen (Fig. 6), demonstrating the efficacy and specificity of the antisense approach. Antisense ODNs targeted against VEGF exhibited a similar effect. After 72 h in culture, an extensive vascular network was visible in control (Fig. 5D) and sense VEGF ODN-treated lung explants (Fig. 5E). Similar to antisense HIF-1{alpha} ODN-treated lung explants, large areas of antisense VEGF ODN-treated lung explants were devoid of vessels (Fig. 5F). The inhibitory effect of antisense VEGF ODN on vascularization was still visible after 96 h in culture (Fig. 5, G–I, for control, sense, and antisense VEGF ODN-treated lung explants, respectively). To determine the specificity of the antisense VEGF knockdown, we tested whether exogenous VEGF could overcome the antisense VEGF ODN inhibitory effect on vascularization (Fig. 7). Addition of recombinant VEGF to lung explant cultures significantly ameliorated the inhibitory action of antisense VEGF ODN on vascular development (Fig. 7 C and G). Incubation of antisense HIF-1{alpha} ODN in the presence of recombinant VEGF only partially overcame the antisense HIF-1{alpha} ODN inhibition on vascularization (Fig. 7K). Addition of recombinant VEGF to control lung explant cultures did not significantly enhance vascularization (Fig. 7, D, H, L). These results show that antisense knockdown of either HIF-1{alpha} or VEGF inhibits vascular development in lung explants cultured at 3% oxygen. Vascularization was more affected by antisense HIF-1{alpha} ODN than antisense VEGF ODN treatment. In addition, exogenous VEGF did abolish the inhibition of vascular development caused by antisense VEGF ODN, but not by antisense HIF-1{alpha} ODN treatment. These findings may be explained by the position of both factors in the hypoxic signaling cascade leading to vascular development. HIF-1{alpha} is the key player and functions upstream in this cascade, regulating VEGF and numerous other genes (62).



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  		Fig. 5. Antisense inhibition of hypoxia-inducible factor (HIF)-1{alpha} and VEGF reduces pulmonary vascularization in vitro. Tie-LacZ expression in E11.5 lung explants after 48 (A–C), 72 (D–F), and 96 (G–I) h at 3% oxygen. Explants treated with antisense oligonucleotides (ODNs) targeted against HIF-1{alpha} (C) or VEGF (F and I) demonstrated abnormal vessel formation compared with sense-treated (HIF-1{alpha}, B; VEGF, E and H) or control (A, D, G) explants. Large areas without vessels were seen in antisense ODN-treated explants (arrows in C and I), whereas in control and sense ODN-treated explants, the whole explant was covered with vessels (arrowheads in A, B, D, E, G, H). Blue color is X-gal staining in endothelial cells. Bars, 100 µm.

 


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  		Fig. 6. Antisense inhibition of HIF-1{alpha} mRNA abolishes HIF-1{alpha} protein expression. E11.5 lung explants were cultured at 3% oxygen in the presence or absence of sense (S) or antisense (AS) HIF-1{alpha} ODNs. After 48 h, proteins were extracted from explants, and HIF-1{alpha} protein content was analyzed by Western blotting. Rat brain was used as positive (C) control.

 


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  		Fig. 7. Exogenous VEGF restores pulmonary vascularization in antisense VEGF, but not HIF-1{alpha}, ODN-treated explants. Tie-LacZ expression in E11.5 lung explants after 48 (A–D, I–J) and 96 (E–H) h at 3% oxygen. Explants treated with antisense ODNs targeted against HIF-1{alpha} (J) or VEGF (B, F) demonstrated abnormal vessel formation compared with control explants (A, E, I). Addition of 100 µg/ml of recombinant VEGF abolished the inhibitory effect of antisense VEGF ODNs on pulmonary vascularization (C, G). Antisense HIF-1{alpha} ODN-triggered reduction of vascularization was not altered by the addition of VEGF (K). The addition of recombinant VEGF to control explants had no major effect on pulmonary vascularization (D, H, L). Blue color is X-gal staining in endothelial cells. Bars, 100 µm.

 
To investigate the effect of reduced vascular development on epithelial branching morphogenesis, C101-LacZ lung explants were treated with sense and antisense ODNs targeted against HIF-1{alpha} or VEGF. LacZ staining of epithelial airway cells revealed an almost complete abrogation of epithelial branching morphogenesis in antisense HIF-1{alpha} ODN-treated lung explants (Fig. 8, C and F). After 72 h in culture, LacZ staining was observed in the trachea, two main bronchi, and subsequent branches of control, sense, and antisense HIF-1{alpha} ODN-treated lung explants. No obvious differences were observed between control (Fig. 8A) and sense HIF-1{alpha} ODN-treated lung explants (Fig. 8B). Conversely, antisense HIF-1{alpha} ODN-treated lung explants showed dramatically reduced epithelial branching with abnormal, blunt tips of distal branches (Fig. 8C). LacZ staining of explants cultured for 96 h showed that epithelial branching morphogenesis in control (Fig. 8D) and sense (Fig. 8E) HIF-1{alpha} ODN-treated lung explants had progressed toward a complex network of fine epithelial branches that had extended and rebranched multiple times in culture. In contrast, antisense HIF-1{alpha} ODN-treated lung explants demonstrated a complete stop in branching morphogenesis (Fig. 8F). Treatment of explants with antisense ODNs targeted against VEGF had similar results, although branching in these explants, after 72 h culture at 3% oxygen, was less affected compared with treatment with antisense HIF-1{alpha} ODNs (Fig. 8, G and H, for control and antisense VEGF ODN-treated lung explants, respectively). These results suggest that inhibition of pulmonary vascular development results in a dramatic reduction in epithelial branching morphogenesis, indicating that in the context of epithelial-mesenchymal tissue interactions, the vascular network within the mesenchyme is a key factor for epithelial branching morphogenesis.



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  		Fig. 8. Antisense inhibition of pulmonary vascular development decreases epithelial branching morphogenesis. C101-LacZ expression in E11.5 lung explants after 72 (A–C, G, H) and 96 (D–F) h at 3% oxygen. Explants treated with antisense ODNs targeted against HIF-1{alpha} (C and F) or VEGF (H) demonstrated an almost complete stop of epithelial branching morphogenesis compared with sense HIF-1{alpha} ODN-treated (B and E) or control (A, D, G) explants. Blue color is X-gal staining in airway epithelial cells. Bars, 100 µm.

 

   DISCUSSION
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In this study, we demonstrate that a low oxygen environment (3% oxygen) stimulates pulmonary vascular and epithelial branching morphogenesis in vitro. Increased epithelial branching was accompanied by increased SP-C expression, implying that low oxygen maintains an appropriate epithelial differentiation pattern. However, the expression of FGF-10 and Bmp4, two critical signaling components for proper distal epithelial branching (49), was oxygen independent. Low oxygen stimulated vascularization, and this was accompanied by a significant increase in PECAM-1 expression. Also, VEGF expression was significantly increased by low oxygen. Similar observations have been reported for another branching organ, the kidney (68). Low oxygen stimulated tubule formation and ureteric bud growth from E14.5 metanephroi in vitro. Moreover, low oxygen exposure of metanephroi increased the number of Vegfr (Flt-1 and Flk-1)-positive endothelial cells and induced a 10-fold increase in VEGF mRNA (68). These data highlight the importance of a low-oxygen environment for proper development of branched organs. Moreover, we show that the inhibition of vascularization in vitro results in a dramatic decrease in epithelial branching morphogenesis, suggesting that pulmonary vascular development is rate limiting for epithelial branching morphogenesis.

The enhancement of lung epithelial branching morphogenesis by low oxygen has been reported by Gebb and Jones (24). They showed increased terminal branching and cellular proliferation in fetal rat lung explants that were cultured at 3% oxygen compared with 21% oxygen (24). Additionally, they reported that low oxygen suppressed the activity of metalloproteinases, zinc-dependent enzymes that modify the extracellular matrix (ECM) structure and function, resulting in the accumulation of specific ECM components, including tenascin-C, which has been shown to influence lung branching (24). Wilborn and coworkers (70) reported a loss of morphology in E12.5 mouse lung explants after 48-h culturing at 10% oxygen. It should be mentioned that Wilborn and coworkers did not use serum in their cultures, whereas in the present study we used 10% FCS. It is plausible that FCS contains factors needed for the survival of the explants at low oxygen. Human fetal lung tissues have been shown to differentiate spontaneously in an atmosphere of 20% oxygen, but the morphological differentiation and SP-A expression disappeared when the tissues were maintained at 1% oxygen (3). Also, the low oxygen effect on morphological differentiation was rapidly reversed when tissues were exposed to 20% oxygen after 5 days at 1% oxygen (3). These findings led the authors to conclude that oxygen plays an important permissive role in the spontaneous differentiation (and SP-A expression) of human fetal lung in vitro. We observed that increased airway branching in a low-oxygen environment was associated with proper terminal differentiation (SP-C expression). Whether the discrepant findings are due to species differences remains to be established.

In Drosophila, hypoxia greatly enhances the branching of terminal tubules during tracheal development (35). Although Bnl, the fly homolog for FGF, was identified as the critical signal in this hypoxic response, no oxygen-dependent differences in the expression of FGF-10 and its receptor Fgfr2 were observed in the present study. The real-time PCR assay used in the current study does not differentiate between the Fgfr2 splice isoforms Fgfr2-IIIb and Fgfr2-IIIc. Thus it is possible that different oxygen tensions trigger Fgfr2 isoform-specific effects. Fgfr2-IIIb has been demonstrated to be important in early murine lung development (5, 11, 15, 53). Another possible mediator of hypoxia-induced branching morphogenesis is insulin-like growth factor (IGF)-I. It has been shown that hypoxia significantly increases both IGF-I and IGF-I type 1 receptor (Igfr-I) mRNA in the neonatal rat lung (51). Furthermore, Han and Amar (29) showed that immunoinhibition of Igfr-I in human fetal lung explants resulted in reduced branching as well as a loss of endothelial cells. They concluded that IGFs via Igfr-I affect lung development, most likely by acting as endothelial survival factors (29). Because a low-oxygen environment stimulates both components of this signaling pathway, it may account for the increased vascularization and subsequent epithelial branching seen in the present study.

Numerous studies have provided evidence that VEGF acts as a potent inducer of endothelial cell growth and that hypoxia is one of its key stimuli (18, 52). In this study, we show that VRGF mRNA expression is significantly upregulated in lung explants cultured at 3% oxygen. Initially, low oxygen also increased the number of Vegfr2/Flk-1 transcripts in the explants. Similar findings have been reported for VEGF expression in human (2) and rat (24) fetal lung explants cultured at low oxygen. VEGF is indispensable for embryonic development since it was shown that even haploinsufficiency of VEGF is enough to cause embryonic lethality (10, 19). In the embryonic lung, VEGF transcripts are mainly detected in the lung epithelium, and its expression increases with advancing gestation and remains high in the adult lung (8, 28, 30). VEGF signals via two endothelial specific, high-affinity tyrosine kinase receptors, Vegfr1 (Flt-1) and Vegfr2 (Flk-1) (20). During lung development, both receptors are strongly expressed in the embryonic lung endothelium (8). Although alveolar epithelium expresses Vegfr2/Flk-1 (40, 55), exogenous VEGF did not directly induce fetal type II cell proliferation or surfactant protein production (55). The expression of VEGF and the two VEGF receptors in adjacent tissues suggest a regulatory signaling loop between the two tissue components during lung development. This suggestion is supported by the findings of Gebb and Shannon (25), who showed that the expression of endothelial Vegfr2/Flk-1 within the mesenchyme required the presence of pulmonary epithelium. As far as we know, no oxygen-dependent upregulation of Vegfr2/Flk-1 expression in the lung has been reported.

In the present study, we observed that inhibition of vascularization with antisense ODNs targeted against either HIF-1{alpha} or VEGF reduced epithelial branching morphogenesis. We chose to target HIF-1{alpha} and VEGF because both are key factors in the hypoxic response and in vascular development. HIF-1{alpha} is upregulated in response to hypoxia and in turn upregulates the expression of oxygen-sensitive target genes such as VEGF (48, 61). HIF-1{alpha} is expressed in the fetal lung (45), whereas HIF-1{alpha}-deficient mice die in utero and exhibit severe vascularization defects (33). We found that both the antisense knockdown of HIF-1{alpha} and VEGF reduced pulmonary vascular development, although the vascular reduction was more severe in explants treated with antisense HIF-1{alpha} ODN than with antisense VEGF ODN. The latter is not surprising because HIF-1{alpha} affects many more genes than VEGF alone (62). The observation that antisense inhibition of vascularization almost completely abrogated epithelial branching morphogenesis suggests that vascular development is essential for airway branching. Several studies have recently highlighted the importance of endothelial cells for developing organs (38). Flk-1-deficient mice die early in embryogenesis due to a lack of blood vessel formation (60). When Flk-1-deficient livers were cultured, initial epithelial layers formed, but subsequent migration of liver epithelial cells into the surrounding septum transversum failed (47). In vitro and in vivo experiments have shown that endothelial cells induce essential steps in pancreas formation, specifically with respect to endocrine pancreatic differentiation (37). Moreover, there is evidence that distal airway branching and vascular formation are linked during pulmonary development. Inhibition of neovascularization in an embryonic lung allograft model with EMAPII, an antiangiogenic protein, resulted in an arrest of airway epithelial morphogenesis at the canalicular stage of lung development (59). Similarly, lungs of mice that lack the heparin sulfate-bound VEGF isoforms 164 and 188 and only express freely diffusible VEGF 120 showed a decrease in peripheral vascular development with fewer air-blood barriers and delayed air space formation (21). Likewise, neonatal mice treated with a soluble decoy receptor for Vegfr1 to block endogenous VEGF signaling exhibited a dramatic decrease in body and organ growth and died within 4–6 days after birth (26). The lungs of these mice were immature with simplification of the alveolar region and a decrease in Flk-1 expression (26). Inhibition of Vegfr signaling using the Vegfr blocker Su-5416 either before or after birth also resulted in reduced pulmonary vascularization and alveolarization (34, 41). On the basis of published results and the data presented in this study, we conclude that pulmonary vascular development has a rate-limiting role in epithelial branching morphogenesis.

The aforementioned conclusion does not exclude the finding that pulmonary vascular development depends on signals from the lung epithelium for its survival (25). One driving force behind endothelial proliferation and guidance is clearly epithelial-derived VEGF. Reciprocal paracrine VEGF signaling has been shown to be important for the formation and vascularization of pancreatic islets (39). In the lung, Raoul and coworkers (55) found that the stimulatory effect of VEGF on fetal type II cells was not direct but was exerted indirectly through reciprocal paracrine interactions, most likely involving mesenchymal and endothelial cells. Hence, a regulatory loop is established in which epithelial-derived VEGF induces vascular development upon which the endothelium signals back, either directly or indirectly via the mesenchymal compartment, to stimulate epithelial branching and differentiation.


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 ABSTRACT
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This work was supported by the Sophia Foundation for Medical Research (SSWO 342, M. van Tuyl) and Canadian Institutes of Health Research (FRN-15273, M. Post).


   ACKNOWLEDGMENTS
 
The authors thank Irene Tseu for technical assistance, Pooja Agarwal for advice on whole mount in situ hybridization, and Angie Griffin for animal handling and care. Martin Post is the holder of a Canadian Research Chair (tier 1) in Fetal, Neonatal, and Maternal Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Post, Program in Lung Biology Research, Hospital for Sick Children Research Institute, 555 Univ. Ave., Toronto, Ontario M5G1X8, Canada (E-mail: martin.post{at}sickkids.ca)

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


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