Fig. 2.

Deletion of Pak1 attenuates hypoxia-induced retinal neovascularization. (A) WT and Pak1^−/− mice pups after exposure to 75% oxygen from P7 to P12 were returned to normal air to develop relative hypoxia. At P17, the retinas were isolated, stained with isolectin B4, and flat mounts were prepared and examined under a fluorescent microscope. Retinal vascularization is shown in the first column. Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images in the first column as viewed under 10× magnification. Scale bars: 500 μm (left two columns), 200 μm (right column). (B–D) Retinal vasculature (B), neovascularization (C) and avascular area (D) were determined as described in the Materials and Methods. (E) All the conditions were same as in A except that at P15 the retinas were isolated, fixed, and cross-sections were made and stained for immunofluorescence analysis of CD31 and Ki67. The right column shows the higher magnification (as viewed at 40× magnification) of the areas selected by the rectangular boxes in the left column images. (F) All the conditions were the same as in A except that at P15 the retinas were isolated, stained with isolectin B4, and flat mounts were made and examined for filopodia under fluorescent microscope at 40× magnification. (G) Retinal endothelial cells from WT and Pak1^−/− mice were isolated and tested for VEGFA (40 ng/ml)-induced sprouting. The bar graphs represent quantitative analysis of three independent experiments or six retinas. The values represent mean±s.d. *P<0.01 vs WT normoxia; **P<0.01 vs WT hypoxia (one-way ANOVA followed by Tukey's post-hoc test).