PMC

E2F1:P53 interaction down-regulates VEGF promoter activity. (A) Schema of cotransfection of VEGF promoter truncation mutant-Luc reporter constructs with pE2F1 in E2F1^−/− fibroblasts to determine the minimal VEGF promoter that mediates VEGF suppression by E2F1. (B) Schematic representation of cis-elements in the minimal VEGF promoter. The E2F1-binding element at approximately −134 to −127 is labeled. Other known elements in this region include an NF-κB site and four Sp1 sites. (C) Western blotting of E2F1 protein in the WT fibroblasts exposed to normoxia (“N”) or hypoxia (“H”). (D) Representative (Upper) and quantification of (Lower) coimmunoprecipitation. Anti-murine E2F1 antibody immunoprecipitates were immunoblotted with an anti-murine p53 antibody.

E2F1^−/− mice exhibit enhanced angiogenic activity. (A) Serial measurement of blood flow recovery after the induction of surgical HLI in WT (□) and E2F1^−/− (▴) mice, using laser Doppler perfusion imaging (LDPI) (n = 35 per group; ∗, P < 0.05). (B) (Left and Center) Representative histology of newly formed capillaries in ischemic hind limbs [identified by CD31 (red), BrdU (brown), and hematoxylin/eosin triple staining]. (Magnification: ×400.) (Right) Quantification of CD31+ BrdU+ double-positive capillary density (six randomly chosen fields per ischemic limb were averaged; n = 5 ischemic limbs per group) 14 days after HLI. (C) HLI in old (20 month) mice. (Upper) Autoamputation rate at day 42 after HLI. (Lower) Laser Doppler blood flow recovery (data at day 42 were collected from the nonautoamputated mice) (Open bar, WT; gray bar, E2F1^−/−; ∗∗, P < 0.01). (D) (Left) Representative histology of vasculature at day 14 of LLC-1 tumor challenge in WT and E2F1^−/− mice (CD31, brown and hematoxylin/eosin staining). (Original magnification: ×200.) (Right) Quantification of CD31+ capillary density of tumor tissue at day 14 of tumor challenge in WT or E2F1^−/− mice. (Six randomly chosen fields per tumor were averaged; n = 5 tumors per group; ∗∗∗, P < 0.001.) (E) Photograph showing hemorrhagic tumors in E2F1^−/− mice.

E2F1 represses p53-dependent VEGF mRNA expression. (A) Cotransfection of VEGF promoter (2.6 kb)-luciferase plasmid and E2F1 expression plasmid vs. control plasmid into WT, E2F1^−/−, or p53^−/− lung fibroblasts, which were subsequently exposed to normoxia (open bar) or hypoxia (gray bar) for 24 h and assayed for luciferase activity (n = 3 per group; ∗∗, P < 0.01). As shown, E2F1 overexpression repressed VEGF promoter activity in E2F1-null cells and WT cells but not in p53-null cells. (B) Ribonuclease protection assay was performed on primary fibroblasts (WT or E2F1^−/−) under conditions of normoxia (“N”) or hypoxia (“H”) for 24 h after transfection with a control empty plasmid, p53WT, or p53DN plasmid. The β-actin-normalized value of VEGF or Ang-1 mRNA in WT cells under normoxia conditions was arbitrarily designated as 100, based on which the relative values of other treatment groups were then extrapolated. Representative examples (i) and quantitative analyses for VEGF (ii) and Ang-1 (iii) from three independent experiments are shown. As shown in ii, the increased expression of VEGF in E2F-deficient fibroblasts was nullified by overexpression of WT p53. In contrast, expression of Ang-1 (iii) was not p53-dependent. (C) Differential effects of E2F1 and HIF-1α on VEGF and erythropoietin promoter activity. WT fibroblasts were cotransfected with indicated plasmids and exposed to hypoxia for 24 h before the luciferase and β-galactosidase assays (n = 3 per group; ∗∗∗, P < 0.001).

Loss of E2F1 increases VEGF production and VEGF-dependent EC proliferation and angiogenesis. (A) Representative ribonuclease protection assay photograph (Upper) or quantification (Lower). (B) Quantitative real-time RT-PCR (n = 3 per group; ∗∗, P < 0.01) for mRNA expression of hypoxia-inducible genes in primary lung fibroblasts (WT or E2F1^−/−) under conditions of normoxia (“N”) or hypoxia (“H”) for 24 h. (C) Representative immunoblotting for VEGF (Upper) and densitometric quantification of relative VEGF levels (Lower) in the ischemic limbs of mice 14 days after HLI (n = 5 per group; ∗, P < 0.05). (D) ELISA for plasma VEGF levels of mice 14 days after tumor challenge (open bar, vehicle; gray bar, LLC-1 tumor; n = 6 per group; *, P < 0.05). (E) ELISA for hypoxia-induced VEGF secretion in E2F1^−/− and WT primary fibroblasts (open bar, normoxia; gray bar, hypoxia; n = 4 per group; ∗, P < 0.05). (F) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for proliferation of human umbilical vein endothelial cells exposed to 10% FBS/M199 medium supplemented with 20% conditioned medium collected from WT fibroblasts or from E2F1^−/− fibroblasts that were cultured under either normoxic or hypoxic conditions for 24 h (open bar, no antibody; gray bar, anti-VEGF antibody at 10 μg/ml; striped bar, control IgG at 10 μg/ml; the two bars indicated by the two ends of each bracket are compared; ∗∗, P < 0.01; ∗∗∗, P < 0.001). This figure is representative of three MTT assays. The contribution of VEGF overexpression to enhanced blood flow recovery in HLI E2F1^−/− mice was evaluated by siRNA-mediated VEGF gene knockdown in the ischemic limb. (G) Representative example (Upper) and quantification (Lower) of tissue VEGF levels (Western blotting) at day 7 after HLI surgery and repeated (days 1 and 3) multisite siRNA i.m. injection. (H) Serial laser Doppler perfusion imaging measurement of blood flow recovery after HLI surgery plus in vivo VEGF knockdown with siRNA (open bar, WT with control GFP siRNA; gray bar, E2F1^−/− with control GFP siRNA; striped bar, WT with VEGF siRNA; filled bar, E2F1^−/− with VEGF siRNA; n = 12 per group; ∗, P < 0.05).