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Hepatocyte Growth Factor Is Required for Alveologenesis in the Neonatal Rat Sanna Padela, Judy Cabacungan, Samuel Shek, Rosetta Belcastro, Man Yi, Robert P. Jankov, and A. Keith Tanswell Canadian Institutes of Health Research Group in Lung Development; Lung Biology Programme, Hospital for Sick Children Research Institute; Clinical Integrative Biology, Sunnybrook and Women’s Research Institute; and the Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

Rationale: Our core hypothesis is that growth factors that have dysregulated expression during experimental neonatal lung injury are likely to be involved in normal postnatal lung growth and alveologenesis. Objectives: To determine if hepatocyte growth factor (HGF) is upregulated in neonatal lung injury and is essential for postnatal alveologenesis. Methods: A neonatal lung injury, in which there were patchy areas of interstitial thickening with a relative increase in the proportion of epithelial cells, was induced in newborn rats by exposing them to 60% oxygen for 14 days. Air-exposed pups had binding of endogenous HGF to its natural receptor, c-Met, inhibited by the intraperitoneal injection of either neutralizing antibody to HGF, or a truncated soluble c-Met receptor. Measurements and Main Results: The 60% oxygen–mediated lung injury was associated with increased lung mRNAs for hepatocyte growth factor and c-Met, relative to air-exposed control lungs, at Day 7 after birth. After exposure to 60% oxygen, immunoreactive HGF was increased at Days 4 and 7, and immunoreactive c-Met was increased at Day 14. In air-exposed pups, intraperitoneal injections of neutralizing antibody to HGF inhibited DNA synthesis in alveoliforming secondary crests, and reduced the number of alveoli in 6-day-old pups. Intraperitoneal injections of a truncated soluble c-Met receptor inhibited DNA synthesis in secondary crests in 4-dayold air-exposed rat pups. Conclusions: HGF and its c-Met receptor are required for normal postnatal alveolar formation from secondary crests, and are upregulated during 60% oxygen–induced neonatal lung injury. Keywords: bronchopulmonary dysplasia; lung growth; lung injury; morphometry; soluble receptors

Chronic neonatal lung injury, or bronchopulmonary dysplasia (BPD), is a common morbidity in extremely premature human infants (1). Recent studies in premature baboons (2) indicate that oxidant injury alone can produce the pathologic features of BPD as originally described (3). Hallmark pathologic features of human BPD in the current era are an arrest/inhibition of alveolar formation, which normally occurs by an in-growth of secondary crests into the large, simple precursor saccules, and interstitial thickening (4). Studies in premature baboons with pathologic features of BPD have demonstrated that cellular changes in their lungs include a disproportionate increase in

(Received in original form April 12, 2005; accepted in final form June 25, 2005) Correspondence and requests for reprints should be addressed to Dr. A. Keith Tanswell, M.B., Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. E-mail: keith.tanswell@ sickkids.ca This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 172. pp 907–914, 2005 Originally Published in Press as DOI: 10.1164/rccm.200504-567OC on June 30, 2005 Internet address: www.atsjournals.org

epithelial cells (5). In humans, 85% of alveolarization occurs after birth (6). In the rat, alveolarization is entirely a postnatal event (6). Neonatal rats exposed to 60% oxygen for 14 days, in common with human infants having BPD and premature baboons with pathologic features of BPD, have both areas of interstitial thickening and areas of arrested septation with inhibition of DNA synthesis (7). Morphogenesis of the lung is controlled by interactions between two tissue layers: the epithelium and mesenchyme. Many growth factors and cytokines have been shown to act as inductive signals mediating epithelial–mesenchymal interactions, and regulating lung morphogenesis, in utero (8). Only a few growth factors that regulate postnatal alveolar growth have been identified to date, including platelet-derived growth factor AA (PDGF-AA) (9), PDGF-BB (10), and vascular endothelial growth factor (11). There is only indirect evidence to implicate dysregulation of specific growth factors in BPD (12), although animal models suggest roles for a number of growth factors and cytokines, including PDGF-BB (10), vascular endothelial growth factor (13), and bombesin-like peptides (14). One potential mechanism for the observation that baboons with a BPD-like injury have a disproportionate increase in pulmonary epithelial cells (5) is an increased expression of a growth factor that preferentially stimulates growth of epithelial cells over that of fibroblasts. Examples of such growth factors are as follows: fibroblast growth factor 7 (15), also known as keratinocyte growth factor; fibroblast growth factor 10 (16); and hepatocyte growth factor (HGF), also known as scatter factor (17). We hypothesized that any such growth factor upregulated during neonatal lung injury would most likely be expressed under control conditions and play a role in normal postnatal alveologenesis. We reasoned that we could use neonatal lung injury as a tool to identify such factors, as previously described for PDGF-BB (10). We chose to initially screen for HGF expression, because of its involvement in normal lung growth in utero (18), its increased expression after ischemia-reperfusion–mediated lung injury (19), and increasing evidence that it plays a critical role in situations in which the adult lung undergoes new alveolar formation (20–25). As described below, the expression of HGF was significantly upregulated in neonatal rat lung tissue exposed to moderate hyperoxia. Having confirmed upregulation of HGF in neonatal rat lung injury, we then used interventions with neutralizing antibody and soluble receptor, acting as decoys to bind endogenous HGF and prevent it from binding to its natural c-Met receptor, to confirm an essential role for HGF and the c-Met receptor in normal postnatal lung growth and alveologenesis. Some of the results of these studies have been previously reported in the form of an abstract (26).

METHODS In Vivo Interventions Animal experiments were conducted according to Canadian Council for Animal Care guidelines. Approvals were obtained from the Animal

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DNA synthesis, pups received 20 ␮g/g intraperitoneal bromodeoxyuridine (BrdU) 2 hours before being killed (30). Injections were via a 30-gauge needle into the right iliac fossa.

RNA and DNA Analyses RNA was isolated as described by Chomczynski and Sacchi (31). Northern blot analyses were performed using 30 ␮g of RNA. To correct for variations in gel loading and membrane transfer, results were normalized to the ribosomal protein L32 RNA (32), which was unaffected by exposure to 60% oxygen (Figure E1 in the online supplement). DNA content was measured as previously described (29). Details of RNA and DNA measurements are provided in the online supplement. Homogenized tissue pooled from pups in individual litters was used for extractions.

Immunohistochemistry After perfusion fixation under constant inflation, lung sections were immunostained for elastin, BrdU, pancytokeratin, HGF, c-Met (the HGF receptor), or vimentin, using an avidin–biotin–peroxidase complex method (33). Details are provided in the online supplement.

HGF and c-Met Quantitation HGF measurements were by a commercially available ELISA, and c-Met quantitation was by Western blot. Total protein content was measured according to Bradford (34). Details are provided in the online supplement.

Morphometric Analyses Lungs were embedded in paraffin, cut in 5-␮m sections, and stained for elastin to enhance recognition of secondary crests. Images were captured randomly from 10 nonoverlapping fields from each section, with four sections/animal and four animals/group. Mean linear intercepts, alveolar surface areas per unit lung volume, secondary crest volume densities, and tissue fractions for each image were measured as previously described (28). Postfixation lung volumes were measured by water displacement, and total alveolar numbers were calculated as described by Weibel and Gomez (35). Details are provided in the online supplement. Figure 1. Low- (A ) and high-power (B ) micrographs to demonstrate epithelial cells, identified by cytokeratin (brown) staining, in neonatal rat lungs after exposure to air for 14 days. In contrast to areas without interstitial thickening (C ), an apparent increase in the proportion of epithelial cells was observed in areas of interstitial thickening (D ) in the lungs of pups exposed to 60% oxygen for 14 days. Higher-power views of areas without and with interstitial thickening are shown in E and F, respectively. The box in F is shown at higher power in the inset. In contrast to areas with large distal air sacs (G ), mesenchymal cells, identified by vimentin (brown) staining, were almost completely absent in areas of interstitial thickening (H ) in the lungs of pups exposed to 60% oxygen for 14 days. Bars ⫽ 100 ␮m

Data Presentation All values are presented as mean ⫾ SEM. Values for mRNA or protein quantitation were from four separate litters. Within each litter, tissue from four average-sized pups had been pooled. All other values are from four or five average-sized individual pups taken from different litters. One-way analysis of variance was used to determine statistical significance (p ⬍ 0.05), followed by post hoc analysis using Duncan’s multiple-range test when significant differences were found between groups (36).

RESULTS Day 14 of Exposure to Air or 60% Oxygen: Increased Epithelial Cell Population

Care Review committees of the Sunnybrook and Women’s and Hospital for Sick Children’s research institutes. Rat pups (10–12/litter) were exposed to air or 60% oxygen in paired chambers for up to 14 days, as previously described (7, 10, 27, 28). For neutralizing antibody interventions, pups were maintained in air for 6 days, and received intraperitoneal injections of 100 ␮g of either neutralizing goat antihuman HGF IgG, or isotype-specific IgG, in 100 ␮l phosphate-buffered saline (PBS) on Days 3, 4, and 5 of life. For soluble receptor interventions, pups were maintained in air for 4 days and received either PBS (vehicle control) or a soluble receptor in PBS (3 ␮g in 20 ␮l PBS containing 0.1% [wt/vol] bovine serum albumin/g bodyweight) on Day 2. The soluble receptors used were HGF-R/Fc or p75 neurotrophin growth factor receptor (NGF-R)/Fc as a control to exclude chimeric proteinmediated nonspecific effects (29). For assessment of cells undergoing

When compared with air-exposed rat pups, pups exposed to 60% oxygen for 14 days had no obvious qualitative difference in the relative proportions of epithelial cells or mesenchymal cells, as assessed by pancytokeratin and vimentin immunoreactivity, respectively, in those lung regions characterized by either large distal airspaces or absence of interstitial thickening (Figure 1). These areas have previously been shown to inhibit lung cell growth (7). However, in those regions of interstitial thickening, previously shown to have active DNA synthesis (7), there was an apparent relative increase of epithelial cells, as previously reported for a baboon model of BPD (5), and an almost complete absence of mesenchymal cells (Figure 1).

Padela, Cabacungan, Shek, et al.: Hepatocyte Growth Factor and Alveologenesis

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exposed pups (Figure 4). Consistent with immunohistochemistry findings, quantitative assessment of lung HGF content, by ELISA, demonstrated significant increases of lung HGF after 4 and 7 days of exposure to 60% oxygen, compared with samples from air-exposed pups (Figure 5A). Rat lung c-Met protein, as assessed by Western blot, was observed as a single 50-kD band (Figure E5), as reported in mice and humans (39, 40) and, in keeping with the appearance by immunohistochemistry, significantly increased after a 14-day exposure to 60% oxygen, when compared with samples from air-exposed pups (Figure 5B). Early Postnatal Alveologenesis: Effect of Anti-HGF IgG

Figure 2. Northern blots revealed four hepatocyte growth factor (HGF) mRNA transcripts of 6, 3, 1.5, and 0.4 kb, of which the major transcript was at 6 kb, which was used for quantitation. Northern blots revealed a single mRNA transcript of 9 kb for c-Met, the HGF receptor. After normalization to L32 mRNA, significant increases in HGF (A ) and c-Met (B ) mRNAs were observed after a 7-day exposure to 60% oxygen (black bars), when compared with air-exposed samples (white bars). *p ⬍ 0.05 by one-way analysis of variance (ANOVA) compared with lung mRNA from animals exposed to air for the same period. Lungs from four average-sized pups in each litter of 10 to 12 pups were pooled. Values are means ⫾ SEM for five (HGF) or four (c-Met) litters in each group.

Exposure to Air or 60% Oxygen: HGF and c-Met mRNA Expression

We observed four HGF mRNA transcripts at 6, 3, 1.5, and 0.4 kb (Figure E2), as reported by others (37). The dominant transcript was at 6 kb, which we used for quantitation. Rat lung c-Met mRNA showed a single transcript at 9 kb (Figure E3), as reported by others (38). When normalized to L32 mRNA, both lung HGF (Figure 2A) and c-Met (Figure 2B) mRNAs were significantly upregulated after 7 days of exposure to 60% oxygen, when compared with mRNAs from pups exposed to air. Exposure to Air or 60% Oxygen: HGF and c-Met Protein Expression

Consistent with the HGF mRNA findings, an apparent increase in immunoreactive HGF, as assessed by immunohistochemistry, was observed on Days 4 and 7 of exposure to 60% oxygen, when compared with sections from air-exposed pups (Figure 3). In contrast to mRNA findings, immunohistochemistry revealed an apparent increase in immunoreactive c-Met only after a 14-day exposure to 60% oxygen, when compared with sections from air-

To determine whether the expression of HGF in the air-exposed neonatal rat lung played a role in normal postnatal lung growth, the HGF ligand was decoyed away from binding to its natural receptor, c-Met, by injected anti-HGF IgG. Pups that received intraperitoneal neutralizing antibodies to HGF on Days 3, 4, and 5 of life had normal bodyweights at Day 6 (Figure 6A), but had significantly reduced lung weights (Figure 6B) and total lung DNA contents (Figure 6C), compared with uninjected control animals or animals that had received an isotype antibody. Postfixation lung volumes were not different between treatment groups (Figure E6A), but, as would be expected from the lung weights and DNA contents, the fraction of lung occupied by tissue was significantly reduced (Figure E6B). Consistent with these findings, relative to that of isotype IgG-injected control pups (Figure 7A) or uninjected pups (Figure 7C), injection of intraperitoneal anti-HGF IgG was associated with an apparent gross simplification of the peripheral lung structure (Figure 7B), resulting in a morphologic appearance similar to that normally present at Day 3 (Figure 7D). After treatment with the antiHGF IgG, there was a reduction in the ratio of secondary crests to tissue (Figure 8A), which did not achieve statistical significance, but the reduction in number of secondary crests per unit area (Figure 8B) was statistically significant. There was also an apparent reduction in the number of secondary crests containing cells undergoing active DNA synthesis, as assessed by BrdU immunohistochemistry (Figure E4), which was confirmed by counts of these crests, when expressed either as a ratio to tissue (Figure 8C) or as a proportion of the total secondary crests (Figure 8D). Mean linear intercepts were increased (Figure 9A), and alveolar surface area (Figure 9B), alveolar density (Figure 9C), and total alveolar number (Figure 9D) were all decreased after treatment with anti-HGF IgG. The impact of the treatment with the anti-HGF IgG was such that values for secondary crests per unit area, secondary crest/tissue ratio, mean linear intercept, alveolar surface area, and alveolar density on Day 6 of life, after treatment, were not significantly different from values for untreated pups on Day 3 of life (Table E1). Treatment with anti-HGF IgG had no apparent effect on c-Met expression, as assessed by immunohistochemistry (Figure E7). Early Postnatal Alveologenesis: Effect of Truncated Soluble HGF Receptor

To both confirm a role for the c-Met receptor in early postnatal alveologenesis and to provide a second approach to decoy HGF, pups were injected with a truncated soluble HGF receptor on Day 2 of life. By Day 4 of life, treatment with the truncated soluble HGF receptor had significantly reduced the secondary crest/tissue ratio (Figure 10A), although not the number of secondary crests per unit area (Figure 10B). The number of secondary crests containing cells undergoing active DNA synthesis was reduced, as assessed by BrdU immunohistochemistry (Figure E8), which was confirmed by a significant reduction in both the BrdUpositive secondary crest/tissue ratio (Figure 10C) and the propor-

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Figure 3. Immunohistochemistry for HGF in lung sections from pups at the day of birth (d0), or after 4, 7, 10, or 14 days in air (d4A–d14A) or 60% oxygen (d4O–d14O). As a negative control, sections were immunostained after immunoadsorption of the antibody with the blocking peptide (BP) against which the antibody was raised. Relative to sections from air-exposed pups, there was apparent upregulation of HGF expression (brown stain) after 4 and 7 days of exposure to 60% oxygen. Bar ⫽ 100 ␮m.

Figure 4. Immunohistochemistry for the HGF receptor, c-Met, in lung sections from pups at the day of birth (d0), or after 4, 7, 10, or 14 days in air (d4A–d14A) or 60% oxygen (d4O–d14O). As a negative control, sections were immunostained after immunoadsorption of the antibody with the blocking peptide (BP) against which the antibody was raised. Relative to sections from air-exposed pups, there was apparent upregulation of c-Met expression (brown stain) after 14 days of exposure to 60% oxygen. Bar ⫽ 100 ␮m.

tion of secondary crests that contained cells with BrdU-positive nuclei (Figure 10D). The simplified lung structure evident after treatment with the truncated soluble HGF receptor (Figure E8) was similar to that observed after treatment with the anti-HGF antibody (Figure E4).

gen for normal alveolar type II pneumocytes both in vitro (41) and in vivo (42), and has been implicated in the repair process after lung injury (19–25, 43). Indeed, the severity of outcome in human infants developing BPD has been inversely correlated with the HGF content of their tracheal aspirates (44). Histologic changes in the lungs of neonatal rats exposed to 60% oxygen for 14 days suggest that, in some areas in which there is active cell proliferation, there is a relative overgrowth of epithelial cells, as previously described in a baboon model of BPD (5). The areas of interstitial thickening are not evident after a 7-day

DISCUSSION HGF is a known mesenchyme-derived morphogenic factor regulating fetal lung organogenesis (18). HGF is also a known mito-


Figure 5. (A ) Lung content of HGF, as assessed by ELISA, after 4, 7, 10, or 14 days of exposure of neonatal rat pups to air (white bars) or 60% oxygen (black bars). A significant increase in HGF content was evident after 4 and 7 days of exposure to 60% oxygen. (B ) Western blot analysis for c-Met revealed a single band of 50 kD. The lung content of c-Met was assessed after 4, 7, 10, or 14 days of exposure of neonatal rat pups to air (white bars) or 60% oxygen (black bars). A significant increase in c-Met content was evident after 14 days of exposure to 60% oxygen. *p ⬍ 0.05 by one-way ANOVA compared with lung HGF or c-Met content of animals exposed to air for the same periods. Lungs from four average-sized pups in each litter of 10 to 12 pups were pooled. Values are means ⫾ SEM for four litters in each group.

exposure (7), are of variable degree after a 10-day exposure, and are consistently present after a 14-day exposure, with a significant increase in tissue fraction (28). This is associated with a significantly increased heterogeneity of alveolar diameters (28). We assume, although we have not specifically identified the cell type, that the observed interstitial thickening with an increase in epithelial cells is due to type II pneumocyte hyperplasia. This finding is consistent with an effect mediated by overexpression

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of one or more growth factors that act preferentially on epithelial cells. On the basis of the findings described herein, HGF might mediate this effect, either alone or in concert with other growth factors. In parallel, we noted a reduction in mesenchymal cells in those areas, demonstrating an increase in epithelial cells. It is of interest that HGF has recently been reported to be capable of stimulating myofibroblast apoptosis in another lung injury (45), yet may inhibit epithelial cell apoptosis (24). Our experiments were not specifically designed to assess the role of HGF in 60% oxygen–mediated lung injury. Rather, our objective was to use this lung injury model to identify candidate growth factors that might play a role in normal postnatal lung growth and alveologenesis. We have used this approach previously to identify altered PDGF-BB expression in lung injury, then confirmed its role in normal postnatal alveolar growth by the use of targeted interventions (10). We initially used intraperitoneal injection of an anti-HGF antibody on Days 3, 4, and 5 of life, with animals being killed on Day 6 of life. These time points coincide with the limited period during which there is an increase in alveolar density in the neonatal rat, which occurs between Days 3 and 8 of life (46), making this the optimal window for detecting effects on secondary crest formation. Alveolar formation is essentially complete by Day 21 of life in the rat (47). Injection of the antiHGF antibody, but not an isotype control antibody, resulted in a reduced lung weight and DNA content. There was a parallel simplification of the lung structure, consistent with an arrest/ inhibition of alveolar formation from secondary crests, the cells of which showed reduced DNA synthesis. Reduced new tissue formation was also reflected in the reduced tissue fraction, in the presence of a normal postfixation lung displacement volume, after treatment with anti-HGF antibody. Mean linear intercepts were increased in the pups treated with the anti-HGF antibody, consistent with a failure of secondary crest-mediated alveolar formation from the larger precursor saccules, which was accompanied by a reduced alveolar surface area/unit lung volume. Quantitation of secondary crest formation revealed a reduced secondary crest/tissue ratio after treatment with the anti-HGF antibody, although this did not attain statistical significance, and a significant reduction in secondary crests per unit area. That this was mediated by an inhibition of cell growth in the secondary crests was suggested by a reduction in the number of secondary crests containing cells undergoing active DNA synthesis, relative either to the total tissue or the total number of secondary crests. Morphologic appearance and morphometric analyses suggested that the anti-HGF antibody had arrested alveolarization at, or close to, the level present at the time that treatment with the anti-HGF antibody was initiated. The specificity of antibodies can never be an absolute certainty; therefore, we elected to use a second strategy to decoy HGF, using a truncated soluble receptor to prevent binding of HGF to its endogenous c-Met receptor. Because of the high cost

Figure 6. Rat pups were either untreated (U) or treated with intraperitoneal injections of isotype IgG (I) or anti-HGF IgG (AH) on Days 3, 4, and 5 of life. Treatment with the anti-HGF IgG had no effect on bodyweight (A ), but significantly reduced lung weight (B ) and total lung DNA content (C ) on Day 6 of life, relative to pups that were either uninjected or had received a control isotype IgG. *p ⬍ 0.05 by one-way ANOVA compared with values for pups that were either uninjected or had received a control isotype IgG. Values are means ⫾ SEM for four averagesized pups from different litters of 10 to 12 pups in each group.

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Figure 7. Compared with the appearance of the lungs from Day-6 pups injected with isotype IgG (A ), or uninjected control pups (C ), the lungs of pups that had been injected with anti-HGF IgG (B ) had a simplified lung structure, similar to that of Day-3 pups (D ), consistent with inhibition of alveolar formation. Bar ⫽ 200 ␮m.

Figure 8. Rat pups were either untreated (U) or treated with intraperitoneal injections of isotype IgG (I) or anti-HGF IgG (AH) on Days 3, 4, and 5 of life. Treatment with the anti-HGF IgG had no significant effect at Day 6 on the secondary crest/tissue ratio (A ), but significantly reduced the number of secondary crests/unit area (B ), the bromodeoxyuridine (BrdU)-positive secondary crest/tissue ratio (C ), and the proportion of secondary crests that were BrdU-positive, relative to pups that were either uninjected or had received a control isotype IgG. *p ⬍ 0.05 by one-way ANOVA compared with values for uninjected animals or animals that had received a control isotype IgG. Values are means ⫾ SEM for five (U) or four (I, AH) average-sized pups from different litters.

Figure 9. Rat pups were either untreated (U) or treated with intraperitoneal injections of isotype IgG (I) or anti-HGF IgG (AH) on Days 3, 4, and 5 of life. Treatment with the anti-HGF IgG significantly increased the mean linear intercept (A ), and significantly decreased the alveolar surface area (B ), the number of alveoli/unit area (C ), and the total lung alveolar count (D ) on Day 6 of life, relative to pups that were either uninjected or had received a control isotype IgG. *p ⬍ 0.05 by oneway ANOVA compared with values for uninjected animals or animals that had received a control isotype IgG. Values are means ⫾ SEM for five (U) or four (I, AH) average-sized pups from different litters.


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neonatal lung injury, which will not be simple given that interventions targeted against either PDGF-BB or HGF can arrest/ inhibit alveolar formation, the hallmark of BPD, thus producing the very result that the interventions should prevent. Conflict of Interest Statement : None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

References

Figure 10. Rat pups were either untreated (U) or treated with intraperitoneal injections of truncated soluble nerve growth factor receptor (sN-R) or truncated soluble HGF receptor (sH-R) on Day 2 of life. By Day 4 of life, treatment with the truncated soluble HGF receptor had significantly reduced the secondary crest/tissue ratio (A ), had not significantly reduced the number of secondary crests/unit area (B ), but had significantly reduced both the BrdU-positive secondary crest/tissue ratio (C ) and the proportion of secondary crests that were BrdU-positive (D ), relative to pups that were either uninjected or had received the control truncated soluble nerve growth factor receptor. *p ⬍ 0.05 by one-way ANOVA compared with values for pups that were either uninjected or had received a control truncated soluble receptor. Values are means ⫾ SEM for four average-sized pups from different litters.

of the soluble receptor, we only performed a limited analysis at an earlier time point, with smaller pups, than that used for the neutralizing antibody intervention. As with the neutralizing antibody approach, injection of the truncated soluble HGF receptor inhibited DNA synthesis in alveoli-forming secondary crests, thus confirming a role for both HGF and its c-Met receptor in growth of secondary crests and alveologenesis in the postnatal lung. On the basis of data from previously mentioned studies in adult lung, it is likely that mesenchymal cell–derived HGF acts on epithelial cell c-Met receptors to mediate, in part or in total, epithelial cell proliferation during outgrowth of secondary crests as alveoli are formed. Furthermore, HGF stimulates an angiogenic phenotype in endothelial cells (48), can promote angiogenesis in the lung (49), and, after injury, can recruit endothelial progenitor cells to the lung (50). Given its motogenic activity for endothelial cells (48), it is therefore possible that some of the inhibition of secondary crest formation that we observed with inhibition of HGF binding to its c-Met receptor was due to inhibition of capillary cell migration, a critical initiator of secondary crest formation (6). Our core hypothesis is that growth factors that are dysregulated in neonatal lung injury will be the same growth factors that mediate normal postnatal alveologenesis. This has previously been shown to be true for PDGF-BB (10) and, on the basis of the studies reported above, is also true of HGF. This approach does not, however, specifically define their role in

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