VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung

We used whole lung cultures as a model to study blood vessel formation in v itro and to examine the role that epithelial-mesenchymal interactions play during embryonic pulmonary vascular development. Mouse lungs were isolated at embryonic day 11.5 (E11.5) and cultured for up to 4 days prior to blood vessel analysis. Platelet endothelial cell adhesion molecule-1 (PECAM/CD31) and thrombomodulin (TM/ CD141) immunolocalization demonstrate that vascula r development occurs in lung cultures. The vascular structures identified i n lung cultures first appear as a loosely associated plexus of capillary-li ke structures that with time surround the airways. To investigate the poten tial role of vascular endothelial cell growth factor (VEGF) during pulmonar y neovascularization, we immunolocalized VEGF in embryonic lungs. Our data demonstrate that VEGF is uniformly present in the airway epithelium and the subepithelial matrix of E11.5 lungs. At later time points, E13.5 and E15.5 , VEGF is no longer detected in the proximal airways, but is restricted to the branching tips of airways in the distal lung. RT-PCR analysis reveals t hat VEGF,,, is the predominant isoform expressed in lung cultures. Crafting heparin-bound VEGF(164) beads onto lung explants locally stimulates a mark ed neovascular response within 48 hr in culture. Semi-quantitative RT-PCR r eveals an 18% increase in PECAM mRNA in VEGF(164)-treated whole lung cultur es as compared with untreated cultures. The restricted temporal and spatial expression of VEGF suggests that matrix-associated VEGF links airway branc hing with blood vessel formation by stimulating neovascularization at the l eading edge of branching airways. (C) 2000 Wiley-Liss, Inc.