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Am J Physiol Lung Cell Mol Physiol 290: L1183-L1192, 2006. First published January 20, 2006; doi:10.1152/ajplung.00175.2005
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Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover

S. Majka,1,2 K. Fox,1,3,4 B. McGuire,5 J. Crossno, Jr.,1,3,4 P. McGuire,5 and A. Izzo6

1Department of Medicine, Cardiovascular Pulmonary Research Section, Divisions of 2Cardiology and 3Pulmonary Sciences and Critical Care Medicine, and 4Denver Veterans Administration Medical Center, University of Colorado Health Sciences Center, Denver, Colorado; 5Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico; and 6Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, Colorado

Submitted 19 April 2005 ; accepted in final form 9 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tight regulation of VEGF-A production and signaling is important for the maintenance of lung development and homeostasis. VEGF null mice have provided little insight into the role of VEGF during the later stages of lung morphogenesis. Therefore, we examined the in vitro effects of autocrine and paracrine VEGF-A production and the inhibition of VEGF-A signaling on a Flk-1-negative subset of fetal pulmonary mesenchymal cells (pMC). We hypothesized that VEGF-A receptor signaling regulates turnover of fetal lung mesenchyme in a cell cycle-dependent manner. VEGF receptor blockade with SU-5416 caused cell spreading and decreased proliferation and bcl-2 localization. Nuclear expression of the cell cycle inhibitory protein, p21, was increased with SU-5416 treatment, and p27 was absent. Autocrine VEGF production by pMC resulted in proliferation and p21/p27-dependent contact inhibition. In contrast, exogenous VEGF-A increased cell progression through the cell cycle. Selective activation of Flt by placental growth factor demonstrated the importance of this receptor/kinase in the VEGF-A responsiveness of pMC. The expression and localization of the survival factor bcl-2 was dependent on VEGF. These results provide evidence that VEGF-A plays a critical role in the regulation of fetal pulmonary mesenchymal proliferation, survival, and the subsequent development of normal lung architecture through bcl-2 and p21/p27-dependent cell cycle control.

proliferation; bronchopulmonary dysplasia; SUGEN 5416

BRONCHOPULMONARY DYSPLASIA (BPD) is a chronic disease affecting the lung parenchyma and associated vasculature following treatment of premature newborns with mechanical ventilation and high oxygen (1, 30, 46). BPD is associated with death and long-term morbidity and has been described as a consequence of arrested normal development due to inappropriate signaling arising from oxygen-induced injury (5, 12, 16, 26, 29, 38, 39, 46, 50). Normally the lung reflects the gestational stage of the neonate at the time of delivery. In BPD the formation of both mature alveoli and pulmonary vasculature is impaired (18, 32, 38, 39). Although the specific cellular and molecular mechanisms underlying BPD are incompletely understood, VEGF-A has been shown to be involved in this disease. Levels of VEGF-A are reduced in the tracheal fluid of neonates exposed to hyperoxia, as well as in clinical autopsy material of infants with BPD (7, 15).

Normal lung morphogenesis is regulated by autocrine and paracrine signals exchanged between the developing epithelium and mesenchyme. VEGF-A is produced by developing epithelium and is a critical mediator of both branching morphogenesis and alveolarization. The differential localization and expression of the VEGF-A isoforms during development create a patterning gradient that guides cell proliferation and tissue assembly (3, 14, 25, 42). Increased VEGF expression in the developing epithelium before the pseudoglandular and canalicular stages disrupts branching morphogenesis and results in mesenchymal thinning (17, 53). During the terminal saccular stage of development, the mesenchyme continues to thin, and the blood vessel endothelium aligns in close proximity to the respiratory epithelium, forming a surface for efficient gas exchange (43, 52, 53). At this time the proportion of mesenchyme to epithelium decreases due to fetal breathing movements that cause mesenchymal cell-cycle arrest and apoptosis (22, 43).

In animal models, decreased VEGF-A levels result in thickening of the mesenchyme, while inhibition of VEGF with a blockade of VEGFRII signaling reduces pulmonary arterial density and radial alveolar counts in infant and adult rats (28, 37, 52). Both increased and decreased levels of VEGF in transgenic animals cause apoptosis of mesenchyme (34, 51). Defects in mesenchymal proliferation and apoptosis have been shown to result in lung hypoplasia and respiratory failure (49). Developing pulmonary mesenchymal cells (pMC) express the VEGF receptors Flt-1 (VEGFRI) and Flk-1 (VEGFRII) in a heterogeneous pattern. Flk-1-expressing mesenchyme is associated with endothelial precursors and vasculogenesis in the developing distal lung (2, 3, 9, 20, 22, 23, 44). Flk-1 levels decrease into adulthood, while flt-1 expression is maintained (23, 44). VEGF-A modulates both Flt-1 and Flk-1 receptor expression to maintain tissue homeostasis (22, 23, 41, 48).

The VEGF-dependent mechanisms that regulate pMC turnover and resultant tissue architecture are unknown. However, because VEGF-A is associated with mesenchymal thickening, we hypothesized that alterations in VEGF-A protein levels will modulate fetal pMC turnover in a cell cycle-dependent manner. We used an Flk-1-negative fetal pMC subpopulation cultured using oxygen tensions similar to the fetal environment (3% O2) as our model. VEGF-A regulated progression through the cell cycle. The differential expression of p21, p27, and bcl-2 following VEGF-A treatment or inhibition suggested a role for these cell cycle-related molecules as downstream effectors of VEGF-A in the regulation of pMC proliferation, survival, and apoptosis. These results demonstrate a direct effect of VEGF-A on the turnover of late terminal saccular/early alveolar stage pulmonary mesenchyme.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and Treatment of Fetal Lung Mesenchymal Cells

All procedures were approved by and performed according to the Animal Care and Use Committee guidelines at the University of Colorado Health Sciences Center (UCHSC). Columbia-Rambouillet sheep at 131 days of gestation were killed with a lethal dose of pentobarbital sodium, and the fetal lungs were harvested. The trachea was then cannulated and infused with saline. Isolated lungs were transferred to preequilibrated tissue culture medium ({alpha}-MEM, 20% fetal calf serum, penicillin-streptomycin; Invitrogen Life Technologies, Carlsbad, CA). Explant tissue from the distal lung was sectioned and placed with media on plastic culture dishes for 48 h under normal culture conditions. After 48 h the tissue was removed from the plates, and additional medium was added. Mesenchymal cells, which had migrated from the explant cultures, were visible at this time and required an additional 2 wk of culture before confluence was reached. Three independent cell isolations were performed.

Cell Characterization

pMC at passage 0 (p0, fresh from explant) were expanded and passaged to p2, at which time they were characterized by immunocytochemistry for the detection of smooth muscle {alpha}-actin (SM{alpha}A), platelet-derived growth factor receptor-beta (PDGFR-beta; mesenchymal markers), the absence of desmin (pericyte marker), cytokeratin (epithelial marker), factor 8, and CD34 (endothelial markers) (n = 3). Additionally, semiquantitative PCR was performed to detect VEGF-A, flk-1, and flt-1 mRNA. Phenotype and growth characteristics were documented. Characterization was repeated at p4 or p5 to ensure that no phenotypic switches occurred during the course of experiments.

Experimental Design

pMC from three independent cell preparations at p4–p7 were used for all experiments. All experiments were performed with two or three replicates and repeated with a different isolation of cells to ensure reproducibility. Cells were routinely passaged when they reached 80% confluence.

For all experiments reported here, pMC having reached 50% confluence were cultured at Denver altitude under either fetal oxygen levels (3% O2) or relative hyperoxia (21% O2) in the presence or absence of VEGF-A (100 ng/ml), placental growth factor (PLGF, 10 ng/ml), or SUGEN 5416 (SU-5416, 10 or 25 µM) and corresponding DMSO controls. The SU-5416 concentrations were chosen based upon the maximal inhibition of VEGF-A receptors reported in previous cell studies (19). At this dose other tyrosine kinase receptors retain their function (19). The cells were collected at days 0, 2, and 7 and analyzed for changes in total cell number and cell cycle, apoptosis, and changes in gene/protein expression as described below. The cells were fixed with 4% paraformaldehyde and stored for analyses.

Cell Turnover

After treatment, viable pMC were counted with a hemocytometer and trypan blue exclusion and subsequently stained using the Krishan method to monitor changes in the cell cycle. In brief, 1 x 106 cells were suspended in Krishan stain containing propidium iodide and allowed to stand overnight at 4°C. Data were collected with a FacsCalibur with Cellquest software (Becton Dickenson, San Jose, CA) and analyzed using Summit software (Cytomation, Ft. Collins, CO). Immunohistochemistry (IHC) to detect Ki67 proliferation antigen was also performed. Apoptotic pMC were identified by IHC to detect cleaved caspase 3 (CC3).

Message and Protein Expression Analysis

For determination of mRNA levels of VEGF-A and VEGFRI and II, total RNA was extracted from fetal lung-derived pMC, using the recommended protocol for TRIzol (Invitrogen Life Technologies, Carlsbad, CA). Total RNA was measured spectrophotometrically, and integrity was confirmed by agarose-formaldehyde gel electrophoresis. Reverse transcription was performed with 1 µg of DNase-treated RNA, using oligo dT and the suggested protocol for the Superscript II kit (Invitrogen Life Technologies). The resulting cDNA was added as a template for each subsequent PCR reaction. PCR reactions were carried out using a Perkin Elmer thermocycler under the following parameters: 94°C 5 min 30 cycles of 94°C 30 s, 60°C 30 s, 72°C 30 s, 72°C 15 min. The primers used to generate cDNA were as follows: VEGF-A: 5'-TCACCGCCTCGGCTTGTCACA-3', 5'-TGTAATGACGAAAGTCTGCAG-3' (100, 250, 380 bp); Flt-1: 5'-CTA TAG CAC CAA GAG CGA CGT G-3', 5'-GGC GTT GAG CGG AAT GTA G-3' (550 bp); Flk-1: 5'-GGA GTT TTT GGC ATC ACG GAA GT-3', 5'-GGA AAC AGG TGA GGT AGG CAG AG-3' (600 bp); GAPDH: 5'-TCACCATCTTCCAGGAGC-3', 5'-CTGCTTCACCACCTTCTTGA-3' (650 bp). The reactions were then electrophoresed on 1.2% agarose gels using ethidium bromide or SYBR green for resolution of DNA amplicons. All PCR amplicons were cloned and sequenced to validate the primer pairs. Densitometry was performed using NIH Image analysis, and the experimental amplicons were standardized to the housekeeping gene GAPDH for each sample. PCR was performed using primers for the following genes: VEGFRI (Flt-1), VEGFRII (Flk-1), VEGF-A, and GAPDH. Data were calculated as the means ± SE (n = 2; 3 replicates in each) of the relative integrated density value, which was obtained as area under the curve following Image Quant Analysis. Ovine fetal pulmonary artery endothelial cells were used as controls for the PCR primers.

IHC and Western Blot Analyses

Differences in protein expression were analyzed by IHC and Western blot analysis. IHC was performed on pMC cultured on chamber slides. Subconfluent pMC were fixed in 4% paraformaldehyde, rinsed with PBS, and incubated with primary antibody, either using the recommended protocol for the M.O.M. kit (Vector) or at [5 µg/ml] in phosphate-buffered saline Tween for 16 h at 4°C. After a wash in PBS, secondary antibody was added (1:500) for 30 min at RT. Secondary antibodies were fluorescent, conjugated to Alexa dyes 488 and 594 (Molecular Probes), or biotinylated for use with the ABC/DAB system (Vector) of signal amplification and detection. Following the IHC procedures, we rinsed slides with PBS to remove unbound antibodies, coverslipped them with DAPI-Vectashield (Vector Labs), and viewed them under an Olympus iX71 scope equipped with fluorescence. To quantify the intensity of bcl-2 IHC in the SU-5416-treated groups, we imported digital images into Adobe Photoshop and performed histogram analyses in the red channel, counting 331,000 pixels. Western blot analysis was used to detect semiquantitative changes in protein levels for the various experimental conditions described. Briefly, cell lysates were standardized for protein content (BCA assay; Bio-Rad), and 20 µg of protein were loaded into each well. The membranes were stained with Ponceau S as a secondary means of ensuring equal sample loading. They were then blocked with 5% normal goat serum and inoculated with primary followed by secondary-biotinylated antibody incubated in Tris-buffered salineTween, and then the ABC/ECL detection was performed (Vector, Amersham). Film was scanned on the STORM Imager and densitometry performed using Image Quant. Primary antibodies for procedures described included the following: SM{alpha}A (SM{alpha}A1A4, DAKO); desmin (DE-R11, DAKO); von Willebrand factor (vWF, F8-86; DAKO); PDGFR-beta (clone 28, BD Transduction); BCL, Bax (N-19, P19; Santa Cruz); p21, VEGF-A, Flt-1 (sc397, sc152, sc316; Santa Cruz); p27 (BD 554069); CC3 (Asp175) (no. 9661, Antibody Cell Signaling Technology); and Ki67 (catalog no. RM-9106, Lab Vision NeoMarkers). Controls for each antibody/protein analysis included a matched isotype control and secondary antibody only as well as whole lung lysate as a tissue positive for the specific antigens.

Statistical Analysis

All experiments followed a randomized block design with the use of cells from a least three different animals. Assays were completed from at least two independent experiments consisting of two to six replicates in each. Data are expressed as means ± SE, and significance between groups was determined by one-way ANOVA or Student's t-test using a statistical software package JMP5 (SAS Institute, Cary, NC). Statistical significance was set at P < 0.05, {alpha} = 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and Characterization of Pulmonary Mesenchyme

To examine the direct effects of VEGF-A on pMC, we isolated a Flk-1neg distal lung cell population representative of the late canalicular/early terminal saccular phase of lung development. This is the stage at which supplemental O2 therapy is typically administered in neonates (1, 13, 36). We identified pMC in vivo using IHC to detect SM{alpha}A (Fig. 1, A and B). They also express VEGF-A message (20, 21) and protein, which can be localized to the cell surface (Fig. 1, C and D). IHC was performed on isolated cell populations to confirm the identity of pMC as mesenchyme (45, 47). VEGF-A was localized to the pMC cell surface in vitro but was not detectable by ELISA in the conditioned medium (Fig. 2A). Our data showed pMC expressed mRNA for the 3 VEGF-A isoforms and low levels of Flt-1 protein (Fig. 2, C–E). Low levels of Flt-1 mRNA expression reflected the protein expression (Fig. 2, B, D, and E). Flk-1 (VEGFRII) message was not detected following isolation or in response to VEGF-A treatment. Cultured pMC expressed uniform basal levels of SM{alpha}A, PDGFR-beta, lacked markers of differentiated vasculature, desmin and vWF, as well as cytokeratin (Fig. 2, E–H). These results confirm that the fetal cell isolates were pMC. They also show that our model cell population did not contain endothelial precursor cells and that the subpopulation under study was indeed Flk-1 negative.


Figure 1
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  		Fig. 1. Identification and characterization of pulmonary mesenchyme in situ. Immunohistochemistry (IHC) was performed on sections of paraffin-embedded fetal day 131 lung tissue to detect smooth muscle {alpha}-actin (SM{alpha}A; A, B) or VEGF-A (C, D) using 3,3'-diaminobenzidine (DAB) detection. A, B: SM{alpha}A was localized to the lung mesenchyme, the smooth muscle layers around blood vessels and airways (AW) including the alveolar SMC. C, D: VEGF-A localized to lung mesenchyme, vascular endothelium, and epithelium. Black arrows, mesenchyme; red arrows, endothelium. Magnification: A and C, x40; B and D, x200.

 

Figure 2
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  		Fig. 2. Isolation and characterization of Flk-1neg pulmonary mesenchyme. IHC using fluorescent secondary antibodies or DAB detection was performed to confirm the isolated population of cells as pulmonary mesenchymal cells (pMC). A: VEGF-A localized to fetal day 131 lung mesenchyme in vitro (x200). B: low levels of Flt-1 were localized to the pMC by IHC. C–E: Semiquantitative RT-PCR analysis was performed to detect temporal changes in VEGF-A and Flt-1 gene expression in the presence (VF) or absence (UT) of exogenous VEGF-A. C, E: pMC express mRNA for 3 VEGF isoforms (188, 164, 120). Levels of expression were not affected by VEGF-A treatment. D, E: temporal expression of Flt-1 message following VEGF treatment (P < 0.1). Data were calculated as means ± SE from 2 independent experiments with 3 replicates in each. F, G: IHC analysis confirmed the expression of SM{alpha}A and platelet-derived growth factor receptor (PDGFR)-beta. H: von Willebrand factor (vWF) was not detected. IDV, integrated density values.

 
VEGF-Dependent pMC Proliferation

VEGF receptor blockade decreased pMC proliferation. We treated pMC with SU-5416, VEGF-A receptor (flt-1 and flk-1) blockade, under 3% O2 concentration. SU-5416 treatment significantly decreased the total cell number compared with untreated controls at days 2 and 7 (Fig. 3A). Ki67 expression, which identifies cells active in the cell cycle, was quantitated and showed significantly lower numbers of actively proliferating cells following SU-5416 treatment compared with untreated control cells at days 2 and 7 (Fig. 3B). There was also a reduction in proliferation between the two SU-5416-treated groups at days 2 and 7. Cell cycle analysis was performed to determine whether the SU-5416 response was dependent on growth arrest. VEGF receptor inhibition by 25 µM SU-5416 treatment resulted in a 13% increase of cells arrested in G1 (P < 0.05) compared with untreated controls, with a corresponding 65% decrease in G2 number (P < 0.013) by day 2. Proliferation was decreased significantly in response to SU-5416.


Figure 3
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  		Fig. 3. VEGF-A regulates pMC cell proliferation. pMC were cultured in 3% oxygen and collected at days (d) 0, 2, and 7 following SU-5416, VEGF-A, or placental growth factor (PLGF) treatment. A, B: VEGF-A receptor blockade decreased pMC proliferation. SU-5416 treatment resulted in a significant decrease in total cell numbers compared with untreated control (UT) at d2 (P < 0.0001) and 7 (P < 0.0001). B: this decrease in cell number correlated with decreased Ki67 expression at d2 (P < 0.03) and 7 (P < 0.005). A significant decrease in Ki67 was also detected between SU-5416-treated groups over time (P < 0.0008). The addition of VEGF-A increased pMC proliferation. C: total cell number was not significantly different between untreated control and VEGF-A-treated groups (d7; P < 0.0532). D: however, Ki67 expression was significantly increased at d7 following VEGF-A treatment (P < 0.015). E: PLGF treatment increased cell numbers by d7 (P < 0.037). Data are presented as means ± SE (n = 4). *P < 0.05; **P < 0.01.

 
Exogenous VEGF-A increases pMC proliferation through Flt-1. Overexpression in the lung of VEGF-A in transgenic mice using an epithelial surfactant protein C promoter disrupts branching morphogenesis and mesenchymal thinning (52, 53). The effects of exogenous VEGF-A treatment on pMC cultured under fetal O2 conditions (3%) were therefore examined. There was no significant increase in total cell number when pMC were treated with VEGF (Fig. 3C). No apoptosis was detected using cleaved caspase IHC. Ki67 expression was correlated with the total cell numbers (Fig. 3D). On day 7 Ki67 expression was decreased in untreated control groups compared with the VEGF-A-treated cells, which were actively proliferating (Fig. 3D). These data were further supported by cell cycle analysis, which detected differences in stage of cell cycle between groups

We found that greater numbers of VEGF-treated pMC were actively proliferating compared with controls. Forty-eight hours following VEGF treatment, cells appeared active in all three phases of growth/synthesis, as evidenced by the 7.6% decrease of cells in G1 (P < 0.0021) and 44% and 35% increase in cells in the S (P < 0.0046) and the G2M (P < 0.0046) phases, respectively, compared with controls. On day 7, the untreated control cells were at rest in G1/G2M, while the VEGF-treated cells were progressing from DNA synthesis, S, to G2M. This progression is evident from the 55% increase in cells in S phase and the 39% decrease of cells in G2M phase (P < 0.020) in the VEGF-treated group. The growth arrest of the untreated control cells correlated with decreased Ki67 expression at day 7, likely as a result of confluence. In response to VEGF treatment, no significant changes in VEGF isoforms (Fig. 2, C and E), Flt-1 (Fig. 2, D and E), or Flk-1 message were identified by RT-PCR.

To define a pathway for pMC responsiveness to paracrine VEGF-A, we evaluated the potential role of Flt-1. We repeated the experiments previously outlined substituting PLGF for VEGF-A, because this factor selectively binds Flt-1 (4, 11). The results were comparable to VEGF-A treatment for criteria analyzed (Fig. 3E). There was no significant change in total cell number until day 7. Cell cycle analyses further illustrated that on day 7 the PLGF pMC progression from G1 (P < 0.060) into S (P < 0.04) was 5–6% higher than controls.

VEGF Regulates the Nuclear Expression of the Cell Cycle Modulator p21 by pMC

The cellular expression and distribution of the cell cycle regulatory proteins p21/p27 in control and SU-5416 (25 µM) treated pMC are shown in Fig. 4. When the untreated control cells reached confluence, p27 expression was localized to the nuclei, whereas p21 was detected in the nucleus and cytoplasm (Fig. 4, K and N). In the SU-5416-treated cells, p27 was not detected (Fig. 4G), but p21 was present in the cytoplasm and nuclei of cells (Fig. 4, J and M). VEGF-A-treated cells exhibited the same expression pattern as untreated control cells (Fig. 4, I, L, O). These results suggest that a subset of cells survives SU-5416 treatment by expressing nuclear p21 and downregulating p27 expression, possibly using this as a potential mechanism of survival.


Figure 4
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  		Fig. 4. pMC expression and localization of cell cycle regulatory proteins. A–F: phase contrast and nuclear staining of pMC on d7. Immunohistochemical analyses of cellular expression and distribution of the cell cycle regulatory proteins p21/p27. G, J, M: SU-5416 reduced p21 expression, and p27 was absent. G, H, I: p27 localized to the nucleus of pMC in untreated control and VEGF-A-treated cells. J–O: p21 was present in both the nucleus and cytoplasm. Magnification x100. M–O: enlarged areas.

 
Alterations in the VEGF Levels Decrease Bcl-2 Expression

Analysis of protein expression and localization of bcl-2 (survival-G1 arrest) was performed to examine a possible quantitative change in the protein levels. Bcl-2 staining was more intense in the untreated pMC (Fig. 5, B and F) compared with the VEGF-A (Fig. 5D)- and SU-5416 (Fig. 5H)-treated groups. The SU-5416 treatment decreased bcl-2 intensity 50%. Protein levels of bcl-2 were decreased at 7 days following VEGF-A treatment compared with untreated controls (Fig. 6A). PLGF treatment caused a similar decrease in bcl-2 protein levels at day 7 (Fig. 6B), similar to the decrease in bcl-2 observed following VEGF treatment.


Figure 5
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  		Fig. 5. VEGF-A affects pMC Bcl-2 expression. Photomicrograph (A, C, E, G) images depict the morphology of pMC in the untreated control and VEGF-A- and SU-5416-treated samples. B, D, F, H: Bcl-2 expression was shown by IHC. Bcl-2 reactivity was most intense in the untreated control cells (B, F) and visible to a lesser extent in the VEGF-A (D)- and SU-5416 (H)-treated samples. Magnification x100.

 

Figure 6
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  		Fig. 6. VEGF-A and VEGF-A receptor inhibition decreased Bcl-2 expression in pMC. Western blot analysis was performed to quantitate changes in Bcl-2 expression. Representative blots of bcl-2 expression are shown above corresponding treatments. pMC were cultured in the presence or absence of VEGF-A (A) or PLGF (B) treatment. Bars show the mean IDV ± SE from Western blot analyses (n = 4–6). pMC responded to the addition of VEGF-A and PLGF with decreased bcl-2 on d7 (P < 0.05, P < 0.03). *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The VEGF-dependent mechanisms that influence pMC turnover and resultant lung architecture are unclear; therefore, we examined the effects of VEGF-A signaling on the fetal late terminal saccular/early alveolar stage of lung development using an Flk-1-negative subset of pMC in vitro. Our data demonstrate that pMC isolated from distal lung during the terminal saccular stage responded to changes in VEGF-A via VEGFRI (Flt-1) with alterations in proliferation and cell cycle progression compared with untreated cells (Table 1). These findings support the hypothesis that changes in the level of VEGF-A during the late stages of lung development may contribute to structural changes of the mesenchyme that underlie persistent lung diseases like BPD.


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  		Table 1. Summary of results

 
During lung development, the pulmonary mesenchyme heterogeneously expresses VEGF receptors and can be divided into Flk-1-positive and Flk-1-negative populations (20, 23, 44, 45). Cells expressing Flk-1 are vascular precursors in the distal lung, and the expression of this receptor decreases in adulthood (23, 41, 48). In contrast, Flt-1 is expressed at higher levels than Flk-1 over the entire course of lung development (22, 41, 48). The expression of Flt-1 is maintained into adulthood, in contrast to Flk-1 (41, 48). pMC were isolated from distal fetal ovine lung explants corresponding to late terminal saccular stage, the stage at which supplemental oxygen therapy is administered to premature human neonates (1, 13, 36). The Flk-1neg pMC migrated from the distal lung tissue and formed adherent colonies in culture (21). This in vitro system lacks the structural complexity of lung tissue, including cell-cell interactions as well as paracrine secreted factors; however, its simplicity allowed us to address the effects of VEGF on the specific population of Flt-1-positive pMC. Another consideration with the cell culture system is the inconsistency of VEGF receptor expression over length of time in culture, possibly due to a perceived injury sustained by the cells during isolation and their resulting compensatory survival mechanisms. To control for this, the pMC were isolated on the basis of their properties as migratory mesenchyme, which characteristically involves an autocrine Flt-1 and VEGF-A pathway (6). Importantly, we did not observe any changes in VEGF-A, Flt-1, or Flk-1 mRNA expression over time (Fig. 2, B and D). We are therefore confident that these cells represent a valid model system for addressing our hypothesis.

The effects of exogenous VEGF-A on pMC under fetal oxygen tension (3% O2) were studied. Changes in VEGF-A expression both increase and decrease lung mesenchyme thickness in transgenic mice, although the mechanisms remain unknown (10, 34, 49, 51, 53). In vivo, proper temporal and spatial distribution of VEGF-A is important for the mesenchyme and adjacent interactions with epithelium or endothelium, required for normal lung development (21, 42). In BPD, VEGF-A and Flt-1 levels are decreased (7, 15, 38). Because Flt-1 and VEGF-A are important in pMC survival, this decrease may lead to increased apoptosis and improper tissue morphogenesis (6). Therefore, we initially studied the effects of inhibition of VEGF-A receptor signaling on pMC using SU-5416, which inhibits the VEGF-A receptor tyrosine kinases Flt-1 and Flk-1. VEGF-A receptor inhibition caused a decrease in cell proliferation and density. Cell cycle analysis and low levels of Ki67 expression showed that greater numbers of SU-5416-treated pMC arrested in G1 after 48 h compared with controls.

Cell cycle arrest in response to VEGF receptor blockade was further supported by increased nuclear p21 expression without p27 expression in SU-5416-treated cells. p21 expression is characteristic of apoptosis resistance and is important for cell survival through cell cycle arrest, during which the cells maintain the ability to proliferate and repopulate (24, 27, 31, 33). The expression of p21 independent of p27 has been shown to protect mesenchymal cells from programmed cell death in low-density cell culture (47). Loss of p21 proceeds the onset of apoptosis (37). The expression of p21 and cell cycle arrest, in addition to the large surface area of the cells, was indicative of repair or survival through increased contact with the extracellular matrix (9). Downregulation of the G1 checkpoint regulators p21 and p27, decreases in bcl-2, and subsequent increased apoptosis in response to SU-5416 have previously been demonstrated in tumor cells (54). The pMC expressing p21 were presumably arrested in G1 to promote survival. It is possible that VEGF inhibition selected cells specifically resistant to this particular stress and caused p21-dependent growth arrest. The growth-arrested cells may then remain viable and subsequently proliferate.

Previous studies using transgenic mice demonstrated that VEGF overexpression in the fetal lung disrupts lung morphogenesis. This disruption is characterized by mesenchymal thinning, decreased myofibroblast differentiation, acinar maturation, and sacculation (53). These pathological changes may be attributed to an inappropriate organization of the mesenchymal cell compartment (53). VEGF-A regulates the expression of Flk-1 and Flt-1 and subsequent cell proliferation or survival, resulting in tissue homeostasis (48). We therefore examined the effects of exogenous VEGF-A on pMC. VEGF-A treatment did not affect the total cell number or apoptosis. However, cell cycle analysis and Ki67 expression showed that greater numbers of VEGF-A-treated pMC were actively proliferating compared with confluent contact-inhibited control cells on day 7. The similarity in the proliferative rate of the groups until day 7 suggests that the effects of exogenous VEGF-A were not detectable until day 7 when confluence was reached in the untreated controls. The continued proliferation of VEGF-treated cells beyond the cell density of control pMC suggests a deregulation of contact inhibition.

We evaluated changes in contact inhibition as a function of cell cycle by examining qualitative changes in protein levels of bcl-2 associated with cell survival (22). Both SU-5416 and VEGF-A decreased levels of bcl-2 protein and altered its localization in pMC. Bcl-2 not only delays the onset of apoptosis but functions to arrest cells in G1 phase of the cell cycle and slow the transition between G1 and S phase (8, 40). Increased levels of bcl-2 protein decrease apoptosis in mice exposed to hyperoxia (5, 22, 39). Therefore, VEGF-A is mitogenic in this instance and regulates pMC progression through the cell cycle, likely through decreases in bcl-2. Bcl-2 is also involved in patterning during development. Its expression is pronounced at sites of ectoderm/mesenchymal interaction (35). VEGF-A may therefore regulate cell-cell interactions influencing the two-dimensional organization of pMC in culture. The proliferative responses of pMC to VEGF-A were mimicked by PLGF treatment, which selectively activates the Flt-1 receptor (4, 25). SU-5416 treatment as VEGF receptor blockade may also inhibit to a lesser extent PDGF receptors (PDGFR-{alpha} and -beta). PDGFR-beta is also involved in mesenchymal differentiation and proliferation (6, 45) and is expressed by the pMC. Future studies will require more specific inhibitors of Flt-1 signaling, inhibition of alternate VEGF receptors, and VEGF-A inhibition.

In conclusion, we demonstrated that Flk-1neg pMC responded to VEGF-A inhibition or addition with significant changes in cell proliferation and cell cycle progression. VEGF-A therefore plays a multifunctional role at the levels of both regulation of cell cycle progression through bcl-2 expression as well as the regulation of survival (Table 1). By studying the effects of VEGF on the properties of the pulmonary mesenchyme, we are beginning to understand developmental processes that contribute to BPD, a disease that involves long-term mesenchymal, vascular, and alveolar irregularities, including alveolar hyperplasia, vascular wall thickening, and fibrosis. Because functional mesenchymal remodeling is necessary for normal alveogenesis and vasculogenesis, alterations in the regulation of mesenchyme must likely play a key role in the underlying architectural defects of this tissue. Further studies are necessary to evaluate the mechanisms by which changes in VEGF-A influence pulmonary mesenchymal architecture at later stages of development and growth within the context of the lung parenchyma.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded in part by American Heart Association Grant SDG-0335052N [principal investigator (PI) S. Majka], the Children's Hospital Research Institute of Denver (pilot PI: S. Majka), National Heart, Lung, and Blood Institute Grants HL-68702 and HL-57149 (PI: S. Abman), and the UCHSC Department of Pediatrics, Pulmonary Section.


   ACKNOWLEDGMENTS
 
We thank Drs. Edward C. Dempsey, Vijaya Karoor, James West, and Katherine Young for input and critical review of this manuscript. We thank Drs. Steven Abman, Theresa Grover, Thomas Parker, and Christine Hunt-Peacock for providing ovine lung tissue; Dr. Jay Westcott, Jon Geske (ELISA Technologies), and Patsy Ruegg (IHCtech Histopathology Services Aurora, CO) for VEGF-A ELISAs and input regarding histochemistry and apoptosis studies; Karen Helm and Michael Ashton for expertise performing the cell sorting experiments; the University of Colorado Cancer Center Flow Cytometry Core (supported by National Institutes of Health Grant 5 P30 CA-46934-15); and Drs. Norbert Voelkel, Ivor Douglas, and Neil Markham for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Majka, SON 3928, Mail stop B-133, 4200 E. 9th Ave., Denver, Colorado 80262 (e-mail: Susan.majka{at}uchsc.edu)

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


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