Inhibition of angiogenesis in vivo by Erg siRNA. (A) HUVECs (105) grown on gelatin-coated 6-well plates were transfected with either control siRNA or Erg-specific siRNA (30 nM) for 48 hours. Erg mRNA levels were quantified using RT-PCR, normalized to GAPDH. Erg mRNA expression was significantly reduced after Erg-siRNA treatment, **P < .01. Values are means plus or minus SEM, n = 3. (B) Whole-cell protein extracts were analyzed by SDS-PAGE immunoblotting with antibodies to Erg and tubulin. Erg protein expression was significantly reduced in the Erg-siRNA treated cells, **P < .01. Values are means plus or minus SEM, n = 3. ICAM-2 protein expression was also down-regulated in Erg-siRNA–treated HUVECs, as determined by SDS-PAGE immunoblotting using antibodies to ICAM-2. (C) Matrigel mixture containing siRNA (2 μM) with or without bFGF (80 ng/mL) was injected subcutaneously into C57BL/6 mice. Matrigel plugs were harvested 7 days after implantation, fixed, sectioned, and H&E stained. (i) Matrigel plug without bFGF. (ii) Matrigel plug plus bFGF. (iii) Matrigel plug with bFGF plus Erg siRNA. (iv) Matrigel plug with bFGF plus luciferase control siRNA. Neovessels containing red blood cells (arrows). Original magnification was 10 ×/0.25 Plan objective lens (Olympus). Vessel density was quantified by counting the number of structures containing a lumen and red blood cells. ** indicates P less than .01. Values are means plus or minus SEM, n = 5. (D) Sections from Matrigel plugs treated with siRNA for luciferase (i,iii) or Erg (ii,iv) were stained with antibodies to CD34 (i,ii) or CD45 (iii,iv) and visualized by immunofluorescence confocal microscopy, overlaid with a transmitted light DIC image to enable the vascular structure of the plug to be observed. For CD34 (red), staining is most intense among endothelial cells lining the neovessels (arrows). The pan-leukocyte CD45 antibody (purple) stains isolated cells of the inflammatory infiltrate within the Matrigel matrix and leukocytes inside the lumen of the neovessels (arrows); however. CD45+ cells are also found lining the neovessels, as previously reported.34 Cell nuclei were counterstained with Sytox Green. Inset: IgG-negative controls. Objective lens used was Plan Neofluar 20×/0.5 (Carl Zeiss). Scale bar represents 50 μm. (E) Immunohistochemistry staining with an anti-Erg Ab in the Matrigel plug sections. (i) Endothelial cells lining neovessels express Erg in Luc-siRNA–treated plugs (arrows). (ii) Plugs treated with Erg siRNA show reduced Erg staining. Insert: Control IgG. Original magnification 20×/0.4 Plan N objective lens (Olympus). Scale bar represents 50 μm. (F) (i) Rare apoptotic cells, as shown by anti-Active caspase 3 staining (arrow), are found in Luc-siRNA–treated Matrigel plug samples. (ii) Active Caspase 3–positive ECs are found in Erg siRNA-treated Matrigel plug samples and appear detached from the vessel wall (arrows). *P < .05. Values are means plus or minus SEM n = 3 Erg and n = 5 control. Scale bar represents 50 μm.
