Figure 3.

An increased level of VEGFR-2 signalling contributes to vascular hyperplasia in Vegfr3^iΔEC retinas. (a) Isolectin B4 staining (in green) of Vegfr3^iΔEC retinas after treatment with VEGFR-3- or VEGFR-2-blocking antibodies during P3–P5. Non-specific rat IgG was used as a control. Arrowheads indicate abnormally thick vessels. Scale bar, 100 μm. (b) Statistical analysis showing the percentage vessel area increase in Vegfr3^iΔEC versus wild-type littermate mice in every treatment group (individual experiments; n = 4, 5 and 4 Vegfr3^iΔEC pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively; and 6, 3 and 5 wild-type pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively). (c) qRT-PCR analysis of Vegfr1 gene (also known as Flt1) expression; n = 4 Vegfr3^iΔEC and 3 wild-type pups. In all analyses of the retina, Cre activity was induced for 48h before the mice were killed. ^*P < 0.05, ^***P < 0.001. Error bars, s.e.m. (d) Cultured HUVECs subjected to siRNA-mediated silencing of VEGFR3 expression (VEGFR3 siRNA) and stimulation with VEGF for the indicated times. VEGFR-2 was immunoprecipitated (IP) followed by immunoblotting (IB) for phosphotyrosine (pY) and VEGFR-2. Numbers below the blots indicate relative intensities of pY to VEGFR-2, normalized to control siRNA at the same time point. Note the increased pVEGFR-2 signal at 30 min and 60min (red). Immunoprecipitation and western blot analysis for VEGFR-3 from the same lysates is shown below. Uncropped images of blots are shown in Supplementary Fig. S9b.