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Background: Defective vascular development is a hallmark of bronchopulmonary dysplasia (BPD) of prematurity. Mechanistic regulation of normal and abnormal vascular development in the saccular/alveolar lung remain incompletely understood. To investigate the role of the endothelial cell (EC) in distal lung vascular arborization and alveolarization (the vascular hypothesis), we targeted Delta like 4 (DLL4), an EC Notch ligand, and master regulator of embryonic angiogenesis, hypothesizing that DLL4-dependent angiogenesis is critical for arborization and alveolarization.

Objectives: i) to map DLL4 and angiogenesis signatures from the canalicular to alveolar phase (human/mouse lung development), and ii) to examine the effect of Dll4 (Dll4+/LacZ) haploinsufficiency and EC-specific Dll4-deletion on lung vascular and alveolar development.

Design/Methods: Immunofluorescence staining and confocal microscopy of DLL4 (EC tip cell marker), JAG1 (EC stalk cell marker), and PECAM on mouse and human lung (autopsy), from canalicular to alveolar phase was performed. In Dll4+/LacZ mouse sections, Dll4 expression pattern was assessed with X-gal staining. Vascular development and alveolarization were assessed via PECAM staining, radial alveolar counts (RAC) and mean linear intercepts (Lm). Tamoxifen inducible, EC specific knockout mice (Dll4 Loxp/Loxp; Cdh5-CreERT2) were used to examine DLL4’s alveolar phase role.

RESULTS: DLL4 was expressed in mouse lung from E17 to P28; in human, 23-43wks gestation, with peak expression in alveolar phase, in small arteries and capillaries, in a PECAM-restricted manner. Stalk cell marker (JAG1), and EC Notch activation (NICD) mirrored and approximated EC DLL4 expression in mouse and human. Lung Dll4 (X-gal) expression was confirmed from E17 to P28 using Dll4+/LacZ mice, with prominent expression in the saccular/early alveolar phase and in secondary septa. Dll4 haploinsufficiency (Dll4+/LacZ) resulted in hyper-sprouting of lung vessels (E17), disorganized capillaries (P6), and narrowed-lumen capillaries and smaller intermediate vessels (P14). Dll4+/LacZ mice had defective alveolarization (decreased RAC and increased Lm). Inducible-deletion of EC Dll4 before alveolar phase (P4, P5) disrupted alveolar development in mid-alveolar phase (P14).

CONCLUSIONS:Dll4-dependent angiogenesis regulates lung vascular development. Loss of one or two copies of Dll4 impairs angiogenesis, disrupting alveolarization. Our data evince a direct role for EC-Dll4 in regulating alveolarization and inform Dll4 investigation as a prime candidate for defective vascularization and alveolarization in BPD (bronchopulmonary dysplasia).

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Delta Like 4 (DLL4), an Endothelial Specific NOTCH Ligand is Critical for Lung Vascular Arborization and Alveolarization