Figure 2.

cZNF609 silencing decreases retinal vessel loss and suppresses pathological angiogenesis in vivo (A) Diabetic C57BL/6 mice (8-week old, male) were received an intravitreous injection of scrambled (Scr) shRNA, cZNF609 shRNA, or left untreated (Ctrl). Retinal trypsin digestion was performed to detect acellular capillaries. Acellular capillaries were quantified in 30 random fields per retina and averaged. Red arrows indicated acellular capillaries. Scale bar, 50 μm (n=5, *P<0.05). (B) The mice were infused with Evans blue dye for 2 h. Fluorescence images of flat-mounted retinas and quantification of Evans blue leakage was conducted. Scale bar, 500 μm (n=5, *P<0.05). (C) ELISA assays were conducted to detect the amount of C-reactive protein (CRP), MCP-1, VEGF, interleukin (IL)-1, IL-6, and TNF-α in retinal lysates (n=5, *P<0.05). (D) Retinal vaso-obliteration (VO) and neovascularization of cZNF609 silencing mice and matched control mice at P12 and P17 were shown by staining retinal vasculature using GS lectin-Alexa Flour 549. Avascular area was highlighted using white line. Scale bar: 100 μm. VO area (at P12 and P17) and pathologic angiogenic area (at P17) was statistically analyzed (n=5, *P<0.05). DR, diabetic retinopathy; Ctrl, control; KD, cZNF609 silencing. All data were from at least three independent experiments.