shRNA gene therapy inhibits tumor angiogenesis by downregulating angiogenic factors. (A-D) Illustrated are representative tumor sections prepared 3 weeks following intratumoral injection of either control vector (A), Del1-shRNA (B), VEGF-shRNA (C), or a combination of Del1-shRNA + VEGF-shRNA plasmids (D). (E) Tumor microvessels were stained with an anti-CD31 Ab and counted in blindly chosen random fields to record tumor microvessel density. A significant difference in microvessel counts in tumors treated with Del1-shRNA or VEGF-shRNA compared with control is denoted by "*", and a highly significant difference between Del1-shRNA + VEGF-shRNA versus control by "**". (F) Western blot analysis of Del1 and VEGF expression in tumors 4 days after injection with control vector (lane 1), Del1-shRNA (lane 2), VEGF-shRNA (lane 3), or Del1-shRNA + VEGF-shRNA (lane 4). Tumor homogenates were stained with Abs against Del1 (upper row), VEGF (middle row), and with an anti-β-actin Ab (lower row). The density of each band was measured and normalized to that of β-actin. *Indicates a significant difference at P < 0.05 in the band intensities of Del1 or VEGF between control vector transfected groups, and ** indicates significant difference from Del1-shRNA or VEGF-shRNA treated tumors.