Fig. 8.

Downregulation of cofilin levels blocks hypoxia-induced retinal neovascularization. (A) An equal amount of protein from normoxic and various time periods of hypoxic retinal extracts was analyzed by western blotting for phosphorylated (p)cofilin levels using its phosphospecific antibodies. (B) All the conditions were the same as in A, except that the WT mice pups were injected intravitreally with control, p38MAPKβ or JNK1 siRNA (siControl, sip38MAPKβ and siJNK1, respectively) at P10, P11 and P13, the retinas were isolated. Then, extracts were prepared and analyzed by western blotting for phosphorylated cofilin levels using its phosphospecific antibodies. The blots in A and B were reprobed for cofilin, p38MAPKβ, JNK1, β-tubulin or CDK4 levels for normalization or to show the target and off-target effects of the indicated siRNA. (C) WT mice pups after exposure to 75% oxygen from P7 to P12 were injected intravitreally with control or cofilin siRNA at P12, P13 and P15. At P15 the retinas were isolated and either tissue extracts were prepared and analyzed by western blotting for cofilin, β-tubulin or CDK4 levels (to show the efficacy of the siRNA on its target and off target molecules), or fixed, cut sections were stained for CD31 and cofilin to show the effect of the siRNA on its target molecule level in endothelial cells. Alternatively, at P17 the retinas were isolated, stained with isolectin B4, and flat mounts were prepared and examined under a fluorescent microscope for retinal neovascularization. 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 under 10× magnification. (D–F) Retinal vasculature (D), neovascularization (E) and avascular area (F) were determined as described in the Materials and Methods. (G) All the conditions were the same as in C except that WT mice pups were injected intravitreally with control or cofilin siRNA at P12 and P13, and at P15 the retinas were isolated, stained with isolectin B4, and flat mounts were made and examined for filopodia. (H) All the conditions were the same as in C except that at P15 retinas were isolated, fixed, cross-sections 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. The bar graphs represent quantitative analysis of three experiments with two retinas for each. The values represent mean±s.d. *P<0.01 vs siControl normoxia; **P<0.01 vs siControl hypoxia. Scale bars: 100 µm (10× images), 50 µm (40× images).