Selective deletion of VEGF in type II alveolar epithelial cells. A: Green fluorescent protein (GFP)-proSurfactant Protein C (proSPC) double staining, to determine the efficiency of SPC-promoter driven Cre-recombinase system in type II cells. GFP is stained with fluorescein isothiocyanate (green) and SPC with Cy3 (red). Nuclei are counterstained blue with 4,6-diamidino-2-phenylindole. Expression of GFP indicates cell-specific recombination and is seen in the majority of proSPC-positive Type II cells. Original magnification ×200. B: In situ hybridization shows lack of VEGF mRNA expression in the alveolar wall of SPC-VEGF knockout (KO) animals. One VEGF positive cell in the KO group is to show the “leakiness” of the system. Arrows indicate VEGF-positive cells. Original magnification ×1000. C: VEGF gene expression in lung tissue evaluated by quantitative Real-Time RT-PCR, showing a significantly reduced expression in SPC-VEGF-KO mice (n = 15) in comparison with wild-type animals (n = 12). *P = 0.0002. D: VEGF immunostaining, showing much weaker staining in the alveolar walls in SPC-VEGF KO mice in comparison with wild-type littermates. Nuclei were counterstained with hematoxylin. Arrows indicate VEGF-positive type II cells, arrowheads indicate VEGF-positive macrophages. Original magnification ×1000. E: Quantification of VEGF-positive cells, which was significantly lower in SPC-VEGF-KO mice (n = 3) in comparison with wild-type littermates (n = 3). *P = 0.001.