N-terminal fragments of CgA inhibit angiogenesis in vitro and in vivo. (A) Effect of N-terminal fragments of CgA (CgA1-78 and CgA1-76) on bFGF and VEGF-induced angiogenesis in the RAR assay. Bars represent the number of capillary-like structures emerging from the aorta rings expressed as percentage of the untreated control (mean ± SD). (B) Effect of CgA1-76 on vessel density and tumor growth in the murine WEHI-164 fibrosarcoma model. BALB/c mice (n = 6 per group) were treated intraperitoneally at the indicated time (arrows) after tumor implantation, with the indicated doses of CgA1-76. Tumors were excised and stained with anti-CD31 antibody (mAb MEC 13.3, BD Pharmingen) and AlexaFluor 546 goat anti–rat IgG (arrows, endothelial staining) and with 4,6-diamidino-2-phenylindole (DAPI; nuclear staining). Vessel density and tumor volumes are shown. Vessel density was quantified by counting the number of red spots (CD31⁺) in each field analyzed by fluorescence microscopy (5 fields/section, 3 sections/tumor, 6 tumors/group) using the ImageJ 1.47d software (National Institutes of Health). Each circle represents the average number of vessels/field/section (n = 18). Representative images of CD31 staining (corresponding to 60% area of original fields) are also shown (10× magnification; bar 200 μm). (C-D) Effect of CgA1-76 on tumor growth in the RMA lymphoma and TS/A adenocarcinoma models. Tumor-bearing mice (n = 8-14 per group) were treated intraperitoneally at the indicated time (arrows) after tumor implantation, with the indicated doses of CgA1-76. (A-C) *P < .05; **P < .01; ***P < .001. (A) Two-tailed t test; (B-C) 2-tailed Mann-Whitney test (treated vs untreated).