FIGURE 3.

Knockdown of Gpr126 suppresses angiogenesis in Matrigel plugs and hypoxia-induced retinal neovascularization. A, RNAi efficiency was determined by Western blotting in NIH3T3 cells. B–D, effect of Gpr126 knockdown by mSh1 (mSh) on angiogenesis in Matrigel plugs. Gross photographs of typical plugs are shown, and Matrigel plugs were sectioned and stained with anti-CD31 antibody to indicate neovascularization (B). Quantification of vessel number per high power field (×200) is shown in C. The mRNA levels of Gpr126 in the cells extracted from indicated Matrigel plugs were detected by qRT-PCR (D). E, knockdown of Gpr126 in mouse retina affects hypoxia-induced retinal angiogenesis. Control or Gpr126 lentiviral shRNA was delivered through intravitreal injection into neonatal mice that were then subjected to OIR model. After perfusion with FITC-dextran (green), retinal flat mounts were prepared and analyzed by confocal microscopy. The red fluorescence indicates the lentiviral infection efficiency. Arrows show pathological neovascular tufts. Avascular areas are surrounded by white lines. F, quantification of relative ratio of avascular area versus total retinal area of each retina was determined as described under “Experimental Procedures.” ImageJ software was used for quantification of avascular area. Error bars represent S.E. *, p < 0.05 compared with control.