Loss of PECAM-1 Function Impairs Alveolarization

Abstract

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. 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 air-way morphogenesis and concurrent vascular development (1Burri P.H. McDonald J.A. Lung Growth and Development. Marcel Dekker, Inc., New York1997: 1035Google Scholar). 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 (2Massaro D. Massaro G.D. Am. J. Physiol. 1986; 251: R218-R224PubMed Google Scholar, 3Massaro D. Massaro G.D. Annu. Rev. Physiol. 1986; 58: 73-92Crossref Scopus (134) Google Scholar). 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 (4Kasahara Y. Tuder R.M. Taraseviciene-Stewart L. Le Cras T.D. Abman S. Hirth P.K. Waltenberger J. Voelkel N.F. J. Clin. Investig. 2000; 106: 1311-1319Crossref PubMed Scopus (967) Google Scholar, 5Maniscalco W.M. Watkins R.H. Pryhuber G.S. Bhatt A. Shea C. Huyck H. Am. J. Physiol. 2002; 282: L811-L823Crossref PubMed Scopus (212) Google Scholar). 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 (6Massaro G.D. Massaro D. Nat. Med. 1997; 3: 675-677Crossref PubMed Scopus (509) Google Scholar, 7Massaro G.D. Massaro D. Am. J. Physiol. 2000; 278: L955-L960Crossref PubMed Google Scholar). The completion of alveolarization must necessarily involve the expansion of the vasculature (8Schachtner S.K. Wang Y. Baldwin H.S. Am. J. Respir. Cell Mol. Biol. 2000; 22: 157-165Crossref PubMed Scopus (133) Google Scholar, 9deMello D.E. Sawyer D. Galvin N. Reid L.M. Am. J. Respir. Cell Mol. Biol. 1997; 16: 568-581Crossref PubMed Scopus (249) Google Scholar) 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) 5The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; EC, endothelial cell; PECAM-1, platelet endothelial cell adhesion molecule; RLMEC, rat lung microvascular EC; H&E, hematoxylin and eosin; mAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; PCNA, proliferating cell nuclear antigen. receptor antagonist) (10Jakkula M Le Cras T.D. Gebb S. Hirth K.P. Tuder R.M. Voelkel N.F. Abman S.H. Am. J. Physiol. 2000; 279: L600-L607Crossref PubMed Google Scholar). 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) (11Newman P.J. Newman D.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 953-964Crossref PubMed Scopus (339) Google Scholar, 12Jackson D.E. FEBS Lett. 2003; 540: 7-14Crossref PubMed Scopus (153) Google Scholar, 13Ilan N. Madri J.A. Curr. Opin. Cell Biol. 2003; 15: 515-524Crossref PubMed Scopus (209) Google Scholar). Several studies have demonstrated that PECAM-1 promotes EC migration (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar, 15Gratzinger D. Canosa S. Engelhardt B. Madri J.A. FASEB J. 2003; 17: 1458-1469Crossref PubMed Scopus (70) Google Scholar, 16O'Brien C.D. Cao G Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2004; 287: C1103-C1113Crossref PubMed Scopus (56) Google Scholar), and treatment with anti-PECAM-1 antibody inhibits vessel formation in rodent models of cytokine or tumor-induced angiogenesis (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar, 17DeLisser H.M. Christofidou-Solomidou M. Strieter R.M. Burdick M.D. Robinson C.S. Wexler R.S. Kerr J.S Garlanda C. Merwin J.R. Madri J.A. Albelda S.M. Am. J. Pathol. 1997; 151: 671-677PubMed Google Scholar, 18Zhou Z. Christofidou-Solomidou M. Garlanda C. DeLisser H.M. Angiogenesis. 1999; 3: 181-188Crossref PubMed Scopus (69) Google Scholar). Further, angiogenesis is reduced in PECAM-1-null mice in a model of chronic inflammation (19Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). 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-1-deficient 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 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 (20Bowden R.A. Ding Z.M. Donnachie E.M. Petersen T.K. Michael L.H. Ballantyne C.M. Burns A.R. Circ. Res. 2002; 90: 562-569Crossref PubMed Scopus (96) Google Scholar) 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 (21Korhonen J. Lahtinen I. Halmekyto M. Alhonen L. Janne J. Dumont D. Alitalo K. Blood. 1995; 86: 828-835Crossref Google Scholar). In Vitro Wounding Assay—Wound-induced migration was performed as described previously (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar). 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 AQueous 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 μg 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 H20 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 septae that intersect a perpendicular line drawn from the center of a respiratory bronchiole of the distal acinus (connective tissue septum or pleura) (22Cooney T.P. Thurlbeck W.M. Thorax. 1982; 37: 572-579Crossref PubMed Scopus (189) Google Scholar). 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 ×40 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 were in at least were by Applied as reverse and GAPDH and were from Applied For mouse Tie-1 and reverse was performed using obtained from Inc and the from Three-dimensional H&E tissue sections, every were on an at using MD). images were then into to were then that the of and then each was as a that be and in are in the data were using analysis of are as were were made using the of mAb on Lung studies were done to the of monoclonal PECAM-1, mAb 37 and mAb on RLMEC function in These with rat PECAM-1 K. Christofidou-Solomidou M. O'Brien C.D. J. I. C. Newman P.J. Albelda S.M. H.M. J. 2000; PubMed Scopus Google Scholar), to RLMEC and inhibit tumor-induced angiogenesis (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar). However, migration of RLMEC in a wound assay was inhibited by mAb 62 in a for but was by mAb 37 Further, of these had an on RLMEC proliferation survival 1, and mAb 62 rodents, the first after birth is the critical for alveolarization (1Burri P.H. McDonald J.A. Lung Growth and Development. Marcel Dekker, Inc., New York1997: 1035Google Scholar). to investigate the involvement of PECAM-1 in rat alveolar development, anti-PECAM-1 were every other day to neonatal rat pups from birth to day and lungs were examined on day of life. and normal animals as microscopy of as as mAb showed thinning of the and septal formation with the first of data for In treatment with mAb 62 resulted in alveolar with alveolar demonstrating Electron microscopy of lungs showed alveolar development and normal secondary septation However, mAb animals had large saccular with invaginations of secondary but complete that of secondary formation had but the process had failed to be with these observations was the that the alveolar determined from H&E sections was in mAb animals with untreated rats or rats treated with or mAb 37 30 Further, radial alveolar of alveolarization demonstrated in mAb animals alveolar counts alveolar counts were determined for lungs obtained on day 15. with in a new by mAb the in vivo of of mAb mRNA as an of endothelial cell (21Korhonen J. Lahtinen I. Halmekyto M. Alhonen L. Janne J. Dumont D. Alitalo K. Blood. 1995; 86: 828-835Crossref Google Scholar), were measured on day of using reverse The expression of this endothelial cell with in animals with the formation of new vessels not in our were in PECAM-1 that EC was not by these animals treated with a known of alveolarization D. Massaro G.D. Annu. Rev. Physiol. 1986; 58: 73-92Crossref Scopus (134) Google Scholar), had decreased mRNA on day 15. EC was by mAb 62 and the antibody inhibited in migration but not EC proliferation or survival 1, and these data suggest that the of alveolarization resulted from an inhibition of EC function and not the loss of viable Three-dimensional of mAb 62 on of serial sections, that of the air spaces were seen on sections and We therefore used computer-assisted reconstruction of lung blocks from untreated and mAb and mAb animals to identified by this process were using with alveolar vessels normal and air spaces sections were confirmed to to the by comparison of image to the reconstruction untreated and mAb animals had alveoli and normal a and However, mAb animals showed that alveoli of were tubular the of the tissue examined from the large as as septal formation were These data that more proximal areas of saccular and alveolar development were by mAb EC contribution to remodeling of more proximal during development. of these that for of the be in the data in in mice are viable G.S. H. H. J. J. A. A. B. G. J. 1999; 162: Google Scholar) and an inhibition of angiogenesis (19Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). the that antibody inhibition of PECAM-1 alveolarization in studies were also done to secondary septation was in PECAM-1-deficient mice. lungs from wild type and PECAM-1-null mice were examined on day or day and at 8 after birth with wild type mice on day 1, the lungs of mice were for a with primary alveolar and alveolar day 7, whereas secondary septation was in mice, the process to be in comparison with wild type mice. 8 the in alveolarization between wild type and PECAM-1-null mice were smaller but Using computer-assisted image analysis, the areas of alveolar spaces were measured to alveolarization. We that immediately after birth and and at 8 the alveolar areas were in PECAM-1-deficient mice with wild type animals Further, at each the radial counts were in the PECAM-1-null mice normal alveolarization is characterized by a in the of the alveoli and a increase in radial alveolar these quantitative data provide evidence of an of alveolarization in PECAM-1-null mice. in PECAM-1-null during mAb 62 inhibited rat postnatal alveolarization without EC content. Tie-1 expression in the lungs of wild type and PECAM-1 animals was also assessed to the loss of PECAM-1 EC content. the absence of PECAM-1 was with impaired alveolarization, Tie-1 mRNA in wild type and PECAM-1-null mice were on days and 14 after birth This was confirmed at the by not with this was the that the proliferation of ECs in and medium vessels as as that of cells the ECs of the the walls of the alveoli were in wild type and PECAM-1-null Further, in alveolar cell apoptosis by TUNEL were between wild type and PECAM-1-deficient mice However, migration by ECs isolated from the lungs of neonatal PECAM-1-null mice was reduced with that of lung endothelial cells obtained from wild type animals Cell proliferation was in both cell not These data therefore suggest that in EC a loss of viable ECs, may be a for the of alveolarization in PECAM-1-deficient mice. Studies were performed with rat and mouse neonatal pups to investigate the involvement of PECAM-1 in alveolarization. We that an anti-PECAM-1 antibody that inhibits EC migration but not proliferation or survival in 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. lung ECs from these PECAM-1 mice demonstrated reduced cell by two we showed that the loss of PECAM-1 function postnatal lung development and implicated the importance of the and endothelial function in to alveolar development. In the the vasculature of the lung from two but concurrent D.E. Sawyer D. Galvin N. Reid L.M. Am. J. Respir. Cell Mol. Biol. 1997; 16: 568-581Crossref PubMed Scopus (249) Google Scholar). the capillary a process of in endothelial to the and then into isolated These remodeling to the capillary vascular angiogenesis to the proximal vasculature the and of new vessels from preexisting ones. Subsequent fusion and of these and the initial of the and during the of alveolar there is a increase in the of the result of angiogenesis in these vessels (8Schachtner S.K. Wang Y. Baldwin H.S. Am. J. Respir. Cell Mol. Biol. 2000; 22: 157-165Crossref PubMed Scopus (133) Google Scholar). The of inhibition of angiogenesis on rat neonatal alveolarization was previously by (10Jakkula M Le Cras T.D. Gebb S. Hirth K.P. Tuder R.M. Voelkel N.F. Abman S.H. Am. J. Physiol. 2000; 279: L600-L607Crossref PubMed Google Scholar), that of of angiogenesis fumagillin, and SU5416, a to neonatal animals to alveolar and lung vascular development. These of the importance of EC to alveolarization, must be with and may have on lung growth of anti-angiogenic S. S. A. PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, Res. 1997; PubMed Scopus Google Scholar). VEGF is a known survival factor for endothelial cells N. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. M. N. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, I. A. J. J. Nat. Med. 1995; PubMed Scopus Google Scholar) and also promotes alveolar type cell growth and K. P. M. H. S. Y. B. M. P. K. L. D. P. Nat. Med. 2002; PubMed Google Scholar), that are by A. J. M. N. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the inhibition of alveolarization by might be to the loss of viable ECs impaired alveolar cell and not the result of reduced angiogenesis. blocks growth receptor in to that of L. N. H. Hirth P. G. C. J. Med. PubMed Scopus Google Scholar). In contrast to these with mAb an anti-angiogenic (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar) that inhibits EC function cell without affecting EC survival and proliferation rat alveolarization without reducing EC PECAM-1-null mice have been and are viable G.S. H. H. J. J. A. A. B. G. J. 1999; 162: Google Scholar), that vascular development, in the absence of PECAM-1, is to for development. However, studies have that the loss of PECAM-1 in endothelial to apoptotic C. Christofidou-Solomidou M. M. Newman D.K. C. Albelda S.M. S. Newman P.J. Blood. 2003; PubMed Scopus Google Scholar), decreased in to inflammation A. G.S. S. Blood. PubMed Scopus Google Scholar, S.M. P. A. J. J.A. Christofidou-Solomidou M. Am. J. Respir. Cell Mol. Biol. 2004; PubMed Scopus Google Scholar), and in other A.R. J. 2004; PubMed Scopus Google Scholar), to M. S. Canosa S. M. Graesser D. Madri J.A. Am. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. C. Newman D.K. Newman P.J. Am. J. Physiol. PubMed Scopus Google Scholar) and decreased angiogenesis in in vivo models (19Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). M. The that alveolarization is impaired in PECAM-1-null mice of the of these also the that there may be in the but these are not of a to the of the mice. Studies are therefore to more defects resulting from the loss of antibody inhibits vessel formation in rodent models of cytokine or tumor-induced angiogenesis H.M. Christofidou-Solomidou M. Strieter R.M. Burdick M.D. Robinson C.S. Wexler R.S. Kerr J.S Garlanda C. Merwin J.R. Madri J.A. Albelda S.M. Am. J. Pathol. 1997; 151: 671-677PubMed Google Scholar, 18Zhou Z. Christofidou-Solomidou M. Garlanda C. DeLisser H.M. Angiogenesis. 1999; 3: 181-188Crossref PubMed Scopus (69) Google Scholar) as as the by vessels of in on mice (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar). The from these studies have been confirmed by the that angiogenesis is reduced in PECAM-1-null mice in a model of chronic inflammation (19Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) as as in models of growth and of M. In vitro, expression of PECAM-1 promotes EC (14Cao G. O'Brien C.D. Zhou Z. Sanders S.M. Greenbaum J.N. Makrigiannakis A. DeLisser H.M. Am. J. Physiol. 2002; 282: C1181-C1190Crossref PubMed Scopus (174) Google Scholar, 15Gratzinger D. Canosa S. Engelhardt B. Madri J.A. FASEB J. 2003; 17: 1458-1469Crossref PubMed Scopus (70) Google Scholar), and we that the loss of PECAM-1 from murine EC cell migration our that EC is in both of the model used and is with the role of PECAM-1 as a of EC the importance of vessel formation and remodeling to alveolarization (8Schachtner S.K. Wang Y. Baldwin H.S. Am. J. Respir. Cell Mol. Biol. 2000; 22: 157-165Crossref PubMed Scopus (133) Google Scholar, 9deMello D.E. Sawyer D. Galvin N. Reid L.M. Am. J. Respir. Cell Mol. Biol. 1997; 16: 568-581Crossref PubMed Scopus (249) Google Scholar), the in alveolar formation resulting from the loss of PECAM-1 function may be to angiogenesis to compromises in EC In to in angiogenesis, at least other be PECAM-1 is an molecule that may play a role in vascular D. A. M. A. S. Engelhardt B. Madri J.A. J. Clin. Investig. 2002; PubMed Scopus Google Scholar). the loss of PECAM-1 function might the of and the of an that might have on endothelial cell C. Christofidou-Solomidou M. M. Newman D.K. C. Albelda S.M. S. Newman P.J. Blood. 2003; PubMed Scopus Google Scholar) endothelial apoptosis at in the large vessels of PECAM-1-deficient mice. In contrast to we a of alveolar cell apoptosis and between wild type and PECAM-1-null animals We the that EC apoptosis, by the loss of PECAM-1 function, might to a of alveolarization. However, this were a its on our appears to be at there is evidence that and are in by from the of the vasculature K. H. J. PubMed Scopus Google Scholar, D. PubMed Scopus Google Scholar). of lung endothelial PECAM-1 during alveolarization might that regulate lung and This is by our in rat pups that anti-PECAM-1 antibody treatment had on the of the more proximal with vascular are the of emphysema in 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and bronchopulmonary dysplasia in J. Lung in Marcel Dekker, Inc., New Scholar). the of the vasculature may be of the of these diseases is by animal In emphysema occurs with of alveolar cell apoptosis, after treatment with a VEGF receptor (4Kasahara Y. Tuder R.M. Taraseviciene-Stewart L. Le Cras T.D. Abman S. Hirth P.K. Waltenberger J. Voelkel N.F. J. Clin. Investig. 2000; 106: 1311-1319Crossref PubMed Scopus (967) Google Scholar), and decreased expression of VEGF and its receptor are with development of alveolar in a model of bronchopulmonary dysplasia W.M. Watkins R.H. Pryhuber G.S. Bhatt A. Shea C. Huyck H. Am. J. Physiol. 2002; 282: L811-L823Crossref PubMed Scopus (212) Google Scholar). endothelial cell the loss of EC by this may with the to or lung and in to the development of of lung We are to Massaro for the of the and with