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.
Highlights
Consist of a core of connective tissue flanked on either side by capillary vessels
Using an antibody blocking strategy, we showed that an anti-PECAM-1 antibody that inhibits endothelial cell (EC) migration, but not EC proliferation or survival in vitro, disrupts normal alveolar septation in neonatal rat pups without reducing EC content
Effect of Anti-PECAM-1 Antibody, monoclonal 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
Summary
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 situ cell proliferation was determined by immunohistochemical staining for PCNA, whereas apoptotic cells were identified by TUNEL assay (R&D Systems, Minneapolis, MN). 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. When statistically significant differences were found (p Ͻ 0.05), individual comparisons were made using the Bonferroni/Dunn test
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