Angiogenic defect in Rap1-ECKO mice. (A-B) Rap1 expression in ECs and tissues from Rap1-ECKO mice and control littermates. (A) Genomic PCR shows excision of one rap1a and both rap1b floxed alleles in Tie2-Cre⁰rap1a^f/+rap1b^f/f (Rap1-ECKO) mice. Only nonexcised floxed rap1a and rap1b alleles are present in Tie2-Cre⁰ controls. Inverted image of EtBr-stained gel is shown. (B) Western blots reveal lack of Rap1b and reduction of total Rap1 protein expression in ECs from Tie2-Cre⁺rap1a^+/+rap1b^f/f (Rap1b-ECKO) and a further reduction in Rap1-ECKO mice. (C) Dose-dependent reduction in retinal neoangiogenesis. P7 retinas stained with fluorescent isolectin from Rap1-ECKO and Tie2-Cre⁰ littermate controls were imaged using Nikon Eclipse TE200 inverted fluorescent microscope (Nikon Instruments Inc) and a 4×/0.13 objective and CoolSNAP ES CCD camera (Photometrics). Shown is a representative image of a quarter of each retina; scale bar indicates 500 μm. Lines have been inserted to indicate composite images. Graph represents mean ratio of vascularized (delineated in red) to total (delineated in green) retinal area in mutant mice relative to controls obtained from 6-11 mice. Vascularized area is significantly reduced in Rap1b-ECKO and deletion of additional Rap1a allele (Rap1-ECKO mice) significantly increases the defect. (D) Matrigel plug assay. Vascularization of VEGF-containing Matrigel plugs recovered from Rap1-ECKO mice after 4 days from Matrigel injection is decreased compared with Tie2-Cre⁰ controls, as visualized by decreased redness (bright field microscopy; left panel) and decreased microvessel density (H&E staining; right panel). There is no visible vascularization in BSA-containing Matrigel plugs. Bottom row of H&E stained sections are magnification of boxed areas; scale bars are 100 μm. Arrows indicate large vessels; arrowheads, RBCs-containing microvessels. Representative results (from n = 4) are shown.