Loss of PECAM-1 Function Impairs Alveolarization*

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Lung development represents a carefully coordinated process of airway morphogenesis and concurrent vascular development (1). For humans and rodents, the final or alveolar stage of lung development occurs principally following birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules (2,3). This secondary septation greatly increases alveolar surface area, enabling efficient gas exchange. The septae begin as invaginations from the saccular wall and consist of a core of connective tissue flanked on either side by capillary vessels. In time, there is stretching and thinning of the septae, accompanied by fusion of the septal capillaries. This remodeling ultimately produces alveolar walls composed of endothelia and epithelia with interstitial tissue interposed between the two cell layers. Identifying the mechanisms that underlie the process of alveolarization may help to increase our understanding of lung diseases such as emphysema or bronchopulmonary dysplasia that are characterized by destruction or failure of development of alveoli (4,5). The promise of alveolarization as a therapeutic target is seen in the studies demonstrating that retinoic acid reverses emphysema and rescues failed septation in relevant animal models (6,7).
The completion of alveolarization must necessarily involve the expansion of the vasculature (8,9) a process that, at this stage of lung development, involves the angiogenic sprouting of new vessels from preexisting ones. The importance of the vascular component to alveolar development is demonstrated by the fact that rat alveolarization is inhibited by treatment with anti-angiogenesis agents (e.g. fumagillin, thalidomide, or SU5416, a vascular endothelial growth factor (VEGF) 5 receptor antagonist) (10). However, all of these treatments result in endothelial cell (EC) apoptosis, making the contribution of ECs to alveolarization difficult to interpret. Nevertheless, these data suggest that molecules that regulate EC activity required for angiogenesis are likely to be important for alveolar development.
One of the endothelial-expressed molecules that appears to play a role in angiogenesis, and thus might be important to the process of alveolarization, is platelet endothelial cell adhesion molecule (PECAM-1) (11)(12)(13). Several studies have demonstrated that PECAM-1 promotes EC migration (14 -16), and treatment with anti-PECAM-1 antibody inhibits vessel formation in rodent models of cytokine or tumor-induced angiogenesis (14,17,18). Further, angiogenesis is reduced in PECAM-1-null mice in a model of chronic inflammation (19). Studies were therefore done in rat and mouse neonatal pups to investigate the involvement of PECAM-1 in alveolarization. Using an antibody blocking strategy, we showed that an anti-PECAM-1 antibody that inhibits EC migration, but not EC proliferation or survival in vitro, disrupts normal alveolar septation in neonatal rat pups without reducing EC content. Subsequent studies in PECAM-1-null mice confirmed these initial findings. Specifically, the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Finally, cell migration by lung ECs isolated from PECAM-1deficient mice was decreased in comparison with ECs from the lungs of wild type mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide the first evidence that inhibition of EC function, in contrast to a loss of viable ECs, affects alveolarization

EXPERIMENTAL PROCEDURES
Cells and Antibodies-Rat lung microvascular EC (RLMEC) was cultured in MCDB-31 medium (Invitrogen) supplemented with L-glutamine, hydrocortisone, heparin, endothelial growth factor supplement (Discovery Laboratories, Warrington, PA), and 10% serum. Lung endothelial cells were isolated immunomagnetically from wild type and PECAM-null mice using published protocols (20) and were cultured in EGM-2 MV medium from Cambrex Bio Science Walkersville, Inc (Walkersville, MD). The binding of monoclonal antibody (mAb) 37 and mAb 62 to RLMEC was determined by fluorescence-activated cell sorting analysis using previously described procedures (21).
In Vitro Wounding Assay-Wound-induced migration was performed as described previously (14). Twenty-thousand ECs were added to 24-well tissue culture plates and allowed to grow to confluence. Linear defects were then made in the monolayer. The wounded culture was washed with phosphate-buffered saline and then incubated for 24 h in medium (with 1% serum). Using computer-assisted image analysis and the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD) images were obtained immediately after wounding and then 24 h later, and the distance migrated by cells at the wound edge was determined. For each condition, 3-5 wounds were analyzed.
Cell Proliferation Assay-Cells were cultured for 24 h in 96-well plates, and the number of viable cells was determined using a commercially available non-radioactive colorimetric assay according to the manufacturer's instructions (CellTiter 96 AQ ueous non-radioactive cell proliferation assay, Promega, Madison WI).
In Vitro Cell Death Detection-For the studies of apoptosis, confluent cells were exposed for 5, 24, or 48 h to serum-free medium or complete medium, with or without antibody. Apoptosis was then assessed using the APOPercentage apoptosis assay (Bicolor Ltd., Belfast. Northern Ireland) with the absorbance measured at 530 nm.
Animals-The Institutional Animal Care and Utilization Committees at both The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine approved all animal care procedures. For studies of postnatal alveolarization, rat pups derived from Sprague-Dawley rats (Charles River Breeding Laboratories, North Wilmington, MA) were untreated or injected intraperitoneally with saline, non-immune IgG, or anti-PECAM-1 antibody every other day beginning on day 1. The animals were sacrificed on day 15. Separate cohorts of rat pups were also treated with 0.1 g of dexamethasone subcutaneously from days 1-14, a treatment known to inhibit alveolarization. PECAM-1-null mice, on a C57/Bl6 background, were the kind gift of Steven Albelda (University of Pennsylvania, Philadelphia, PA). Wild type mice, also on a C57/Bl6 background, were obtained from Taconic (Germantown, NY). Lungs were harvested on days 1, 7, and 14 and then at week 8 of life.
Light and Electron Microscopy-Lungs were harvested after inflation to a pressure of 20 cm H 2 0 using 10% formalin (paraffin sections) or 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) (for electron microscopy). Paraffin blocks were sectioned for H&E staining, whereas electron microscopy sections were obtained after post-fixation in 2% osmium tetroxide and embedding in epoxy resin. Sufficient sections for representative serial sampling were produced and reviewed. Mean alveolar areas were measured by computer-assisted image analysis, and radial alveolar counts were determined by counting the number of sep-tae that intersect a perpendicular line drawn from the center of a respiratory bronchiole of the distal acinus (connective tissue septum or pleura) (22). Using paraffin tissue sections, in situ cell proliferation was determined by immunohistochemical staining for PCNA, whereas apoptotic cells were identified by TUNEL assay (R&D Systems, Minneapolis, MN). For the PCNA staining of the endothelial cells of the noncapillary vessels, the percentage of total EC that were PCNA-positive was determined for at least 30 vessels for each mouse. For the quantitation of alveolar cell proliferation, the percentage of total alveolar cells that were PCNA-positive on a ϫ40 field was determined for at least 60 fields for each mouse.
Real-time Reverse Transcription-PCR-For rat Tie-1 quantitative real-time reverse transcription-PCR, relative mRNA expression was assessed using polymerase-activated fluorescent PCR probes providing continuous message quantification during amplification (TaqMan, Applied Biosystems, Foster City, CA). Differences in gene expression were determined by comparing the number of PCR cycles required to achieve a threshold of fluorescent activity above background during the exponential phase of the reaction. Normalization of sample loading was performed by the simultaneous amplification of GAPDH (with its own fluorescent probe) in each well. All reactions were carried out in at least triplicate. Specific primers/probes were synthesized by Applied Biosystem's oligo synthesis service as follows: Tie1 forward primer, 5Ј-GC-CCTTTTAGCCTTGGTGT-3Ј; reverse primer, 5Ј-TTCACCCGATC-CTGACTGGTA-3Ј; and probe, (5Ј-FAM fluorescent-reporter)-AG-CTGCCTACATCGGAGACGCACC-3Ј. GAPDH and specific primers/probes were purchased from Applied Biosystems. For mouse Tie-1 and ␤-actin, reverse transcription-PCR was performed using primer pairs obtained from R&D Systems Inc and the Superscript first-strand synthesis system from Invitrogen.
Computer-generated Three-dimensional Reconstructions-Sixty 5-m-thick H&E tissue sections, taken every 5 m, were imaged on an Axiophot Zeiss microscope at low magnification using Image software (Scion, Fredrick, MD). Serial images were then imported into SoftWoRx software (Applied Precision, Issaquah, WA) to form three-dimensional images. Polygons were then created that outlined the limits of specific structures, and then each polygon was assigned as a volume object that could be viewed and analyzed in three-dimensions. More details are provided in the supplemental data online.
Statistical Analyses-Differences among groups were analyzed using one-way analysis of variance. Results are presented as means Ϯ S.E. When statistically significant differences were found (p Ͻ 0.05), individual comparisons were made using the Bonferroni/Dunn test.

RESULTS
Effect of Anti-PECAM-1 Antibody, mAb 62, on Rat Lung Microvascular Endothelial Cells-Initial studies were done to determine the effects of monoclonal antibodies against human PECAM-1, mAb 37 and mAb 62, on RLMEC function in vitro. These functionally distinct antibodies cross-react with rat PECAM-1 (23), bind to RLMEC (Fig. 1a), and inhibit tumor-induced human angiogenesis (14). However, migration of RLMEC in a wound assay was inhibited by mAb 62 in a dose-dependent manner (data shown for 100 g/ml) but was unaffected by mAb 37 (Fig.  1b). Further, neither of these antibodies had an effect on RLMEC proliferation and/or survival (Fig. 1, c and d).
mAb 62 Inhibits Postnatal Rat Alveolarization-In rodents, the first 2 weeks after birth is the critical period for alveolarization (1). Therefore, to investigate the potential involvement of PECAM-1 in rat alveolar development, anti-PECAM-1 antibodies were administered every other day to neonatal rat pups from birth to day 14, and lungs were examined on day 15 of life. Untreated and normal IgG-treated animals served as controls. By light microscopy of equivalently inflated lungs, control animals, as well as mAb 37-treated animals, showed thinning of the mesenchyme and increasing septal formation with time over the first 2 weeks of life ( Fig. 2a; data shown for IgG). In contrast, treatment with mAb 62 resulted in distorted alveolar architecture, with alveolar sacs demonstrating poorly developed and/or incomplete alveoli. Electron microscopy of control lungs showed appropriate alveolar development and normal secondary septation (Fig. 2b). However, mAb 62-treated animals had large saccular structures with invaginations of secondary crests but few complete alveoli, suggesting that appropriate initiation of secondary crest formation had occurred but the process had failed to be completed. Consistent with these morphological observations was the finding that the mean cross-sectional alveolar area (m 2 ) determined from H&E sections was 50% greater in mAb 62-treated animals when compared with untreated control rats or rats treated with IgG or mAb 37 ( Fig. 2c; 333 Ϯ 30 mm 2 (control) versus 486 Ϯ 84 m 2 (mAb 62); p Ͻ 0.001, n ϭ 4 -8). Further, radial alveolar counts, another index of progressive alveolarization demonstrated significantly fewer intercepts in mAb 62-treated animals ( Table 1).
Endothelial Content Is Unaffected by mAb 62-To understand the possible in vivo mechanism of action of mAb 62, Tie1 mRNA levels, as an index of endothelial cell content (21), were measured on day 15 of life using real time reverse transcription-PCR. The expression of this specific endothelial cell marker increased with age in control animals consistent with the formation of new vessels (data not shown). Significantly, in our studies, no differences were noted in any PECAM-1 antibodytreated group (Fig. 2d), indicating that EC content was not affected by these treatments. As expected, animals treated with dexamethasone, a known inhibitor of alveolarization (3), had significantly decreased Tie1 mRNA content on day 15. As EC content was unaffected by mAb 62 and the antibody inhibited in vitro wound-induced migration (Fig. 1b), but not EC proliferation or survival (Fig. 1, c and d), these data suggest that the disruption of alveolarization resulted from an inhibition of PECAM-1-dependent EC function (migration) and not the loss of viable ECs.
Three-dimensional Reconstructions Reveal Additional Effects of mAb 62 on Airway Architecture-Upon closer examination of serial sections, it became evident that some of the enlarged air spaces were seen on multiple sections and appeared contiguous. We therefore used computer-assisted three-dimensional reconstruction of 300-m lung blocks from untreated controls and mAb 62-and mAb 37-treated animals to further analyze airway morphology (Fig. 3). Objects identified by this process were differentiated using colors, with alveolar structures designated green, blood vessels designated red, normal airways designated gray, and abnormal contiguous air spaces designated yellow. Individual sections were confirmed to contribute to the three-dimensional picture by comparison of image stacks to the three-dimensional reconstruction (Fig. 3). Both untreated controls and mAb 37-treated animals had nor-mal-sized alveoli and normal airway structures (Fig. 3, a and c). However, mAb 62-treated animals showed that although some alveoli of appropriate sizes were present, larger tubular structures spanned the whole length of the tissue block examined (Fig. 3b). Incomplete budding from the large tubules as well as abnormal septal formation were noted. These data indicate that even more proximal areas of saccular and alveolar development were affected by mAb 62, suggesting ongoing EC contribution to remodeling of more proximal airways during development. Movies of these reconstructions that allow for better visualization of the structures can be found in the supplemental data on-line.   FIGURE 3. Three-dimensional volume projected images of models computed from histological serial sections. In volume rendered models, green objects indicate alveoli, and semitransparent gray objects indicate airways or terminal bronchioles. Scale bar, 100 m. a, untreated control lung. In this region, near the pleural surface, alveoli (green) had similar size and shape and were arranged around a connecting terminal bronchiole (gray). An arteriole (red) is visible crossing through part of this tissue volume. b, lung treated with mAb62.
Although several alveoli (green) were observed, larger "tubule" structures (yellow) traversed a relatively large thickness of tissue. Incomplete septum formation was observed along the walls of these structures, indicating initiation of alveolar formation. c, lung treated with mAb37. Alveoli (green) were observed surrounding airway structures (gray), as in the control lung. Impaired Alveolarization in Mice Deficient in PECAM-1-PECAM-1-null mice are viable (24) and demonstrate an inhibition of angiogenesis (19). Given the finding that antibody inhibition of PECAM-1 disturbs alveolarization in rats, studies were also done to determine whether secondary septation was affected in PECAM-1-deficient mice. Appropriately inflated lungs from wild type and PECAM-1-null mice were examined on day 1 or day 7 and at 8 weeks after birth (Fig. 4a). When compared with wild type mice on day 1, the lungs of knock-out mice were notable for a very disorganized architecture with larger primary alveolar sacs and thickened alveolar walls. On day 7, whereas some secondary septation was noted in mutant mice, the process appeared to be retarded in comparison with wild type mice. By 8 weeks, the differences in alveolarization between wild type and PECAM-1-null mice were smaller but still present. Using computer-assisted image analysis, the mean areas of alveolar spaces were measured to assess alveolarization. We found that immediately after birth (days 1 and 7) and at 8 weeks (adult), the mean alveolar areas were significantly greater in PECAM-1deficient mice when compared with wild type animals (Fig. 4b). Further, at each time point, the radial counts were significantly lower in the PECAM-1-null mice (Fig. 4c). As normal alveolarization is characterized by a decrease in the cross-sectional area of the alveoli and a progressive increase in radial alveolar counts, these quantitative data provide evidence of an impairment of alveolarization in PECAM-1-null mice.
Endothelial Content Is Preserved in PECAM-1-null Mice during Alveolarization-As noted above, mAb 62 inhibited rat postnatal alveo-larization without altering EC content. Given this, Tie-1 expression in the lungs of wild type and PECAM-1 knock-out animals was also assessed to determine whether the loss of PECAM-1 affected EC content. Although the absence of PECAM-1 was associated with impaired alveolarization, Tie-1 mRNA levels in wild type and PECAM-1-null mice were comparable on days 1 and 14 after birth (Fig. 5a). This was confirmed at the protein level by immunoblotting (data not shown). Consistent with this was the finding that the proliferation of ECs in small and medium sized vessels (Fig. 5b), as well as that of cells (including the ECs of the microvessels) within the walls of the alveoli (Fig. 5c), were similar in wild type and PECAM-1-null animals. Further, no differences in alveolar cell apoptosis (detected by TUNEL staining) were noted between wild type and PECAM-1-deficient mice (Fig. 5d). However, wound-induced migration by ECs isolated from the lungs of neonatal PECAM-1-null mice was significantly reduced when compared with that of comparable lung endothelial cells obtained from wild type animals (5e). Cell proliferation was similar in both cell types (data not shown). These data therefore suggest that functional impairments in EC motility, rather than a loss of viable ECs, may be a mechanism for the disruption of alveolarization in PECAM-1-deficient mice.

DISCUSSION
Studies were performed with rat and mouse neonatal pups to investigate the involvement of PECAM-1 in alveolarization. We found that an anti-PECAM-1 antibody that inhibits EC migration but not prolifer- ation or survival in vitro disrupts normal alveolar septation in neonatal rat pups without reducing EC content. Further, in PECAM-1-null mice, the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Additionally, lung ECs from these PECAM-1 mutant mice demonstrated reduced cell migration. Thus, by two complimentary approaches, we showed that the loss of PECAM-1 function disturbs postnatal lung development and implicated the importance of the pulmonary vasculature, and endothelial function in particular, to alveolar development.
In the embryo, the vasculature of the lung develops initially from two separate but concurrent processes (9). Peripherally, the capillary network arises through a process of vasculogenesis in which endothelial precursors, recruited to the pulmonary mesenchyme, differentiate and then organize into isolated sinusoidal spaces. These "blood lakes" undergo subsequent remodeling to yield the connected capillary vascular tree. Centrally, angiogenesis gives rise to the proximal precapillary vasculature through the budding and branching of new vessels from preexisting ones. Subsequent fusion and coalescence of these peripheral and central systems completes the initial establishment of the pulmonary circulation. Near term, and particularly during the period of alveolar septation, there is a significant increase in the density of intrapulmonary vasculature, the result of angiogenesis in these peripheral vessels (8).
The effects of inhibition of angiogenesis on rat neonatal alveolarization was studied previously by Jakkula et al. (10), who reported that administration of three antagonists of angiogenesis (thalidomide, fumagillin, and SU5416, a VEGFR-2 inhibitor) to neonatal animals leads to alveolar simplification and attenuated lung vascular development. These studies, although suggestive of the importance of EC to alveolarization, must be interpreted with caution. First, fumagillin and thalidomide may have effects on lung growth independent of their anti-angiogenic activities (25)(26)(27). Second, VEGF is a known survival factor for endothelial cells (28 -30) and also promotes alveolar type 2 cell growth and differentiation (31), effects that are mediated by VEGFR-2 (29). Thus, the inhibition of alveolarization by SU5416 might be due to the loss of viable ECs and/or impaired alveolar epithelial cell maturation and not the direct result of reduced angiogenesis. Lastly, SU5416 blocks platelet-derived growth receptor signaling in addition to that of VEGFR-2 (32). In contrast to these antagonists with potentially multiple and/or undefined effects, mAb 62, an anti-angiogenic reagent (14) that inhibits EC function (i.e. cell migration) without affecting EC survival and proliferation (Fig. 1), compromised rat alveolarization without reducing EC content (Fig. 2).
PECAM-1-null mice have been developed and are viable (24), suggesting that vascular development, in the absence of PECAM-1, is sufficient to allow for adequate embryonic development. However, subsequent studies have shown that the loss of PECAM-1 results in increased endothelial sensitivity to apoptotic stress (33), decreased neutrophil recruitment in response to interleukin-1-driven inflammation (34,35), and in other settings (36), enhanced susceptibility to endotoxic shock (37,38) and decreased angiogenesis in various in vivo models (19). 6 The finding that alveolarization is impaired in PECAM-1-null mice (Fig. 4) provides additional definition of the phenotype of these animals. They also raise the possibility that there may well be earlier effects in the embryo, but these are not of a sufficient magnitude to compromise the viability of the mice. Studies are therefore underway to identify more subtle embryonic defects resulting from the loss of PECAM-1.
Anti-PECAM-1 antibody inhibits vessel formation in rodent models of cytokine or tumor-induced angiogenesis (17,18) as well as the vascularization by human vessels of tumors grown in human skin grafts transplanted on immunodeficient mice (14). The conclusions from these antibody-blocking studies have been confirmed by the finding that angiogenesis is reduced in PECAM-1-null mice in a model of chronic inflammation (19) as well as in models of tumor growth and vascularization of subcutaneous Matrigel implants. 6 In vitro, expression of PECAM-1 promotes EC motility (14,15), and we observed that the loss of PECAM-1 from murine EC impairs cell migration (Fig. 5e). Thus, our finding that EC content is preserved in both of the model systems used (Figs. 2d and 5a) is consistent with the putative role of PECAM-1 as a facilitator of EC migration. Consequently, given the importance of vessel formation and remodeling to alveolarization (8,9), the disturbances in alveolar formation resulting from the loss of PECAM-1 function may be directly related to insufficient and/or inefficient angiogenesis due to compromises in PECAM-1-dependent EC function. In addition to disturbances in angiogenesis, at least three other processes should be considered. First, PECAM-1 is an intercellular/junctional molecule that may play a role in regulating vascular permeability (39). Thus, the loss of PECAM-1 function might enhance the extravasation of serum proteins and the elaboration of an abnormal matrix that might have inhibitory effects on epithelial and/or endothelial cell activity. Second, Gao et al. (33) observed increased endothelial apoptosis at baseline in the large pulmonary vessels of PECAM-1-deficient mice. In contrast to their report, however, we detected a low level of alveolar cell apoptosis and no differences between wild type and PECAM-1-null animals (Fig. 5d). We cannot completely exclude the possibility that enhanced EC apoptosis, induced by the loss of PECAM-1 function, might contribute to a disruption of alveolarization. However, if this were a factor, its effect, based on our data, appears to be modest at best. Last, there is evidence that pancreatic differentiation and liver organogenesis are regulated in part by signals from the endothelium of the vasculature (40,41). Thus, engagement of lung endothelial PECAM-1 during alveolarization might initiate signals that regulate lung epithelial differentiation and airway morphogenesis. This possibility is suggested by our finding in rat pups that anti-PECAM-1 antibody treatment had effects on the architecture of the larger, more proximal conducting airways (Fig. 3).
Alveolar abnormalities with vascular pathologies are the hallmark of emphysema in adults (42) and bronchopulmonary dysplasia in preterm infants (43). That the targeting of the vasculature may well be part of the pathogenesis of these diseases is suggested by animal studies. In rats, emphysema occurs with induction of alveolar cell apoptosis, after treatment with a VEGF receptor antagonist (4), and decreased expression of VEGF and its Flt-1 receptor are associated with abnormal development of alveolar capillaries in a baboon model of bronchopulmonary dysplasia (5). Thus, endothelial cell death and/or the loss of functionally intact EC (as suggested by this report) may interfere with the host response to environmental insults or lung injury and in turn contribute to the development of certain forms of parenchymal lung disease.