Slit3 induces angiogenesis in vitro and ex vivo. (A) Slit3 induced EC tube formation. HUVECs cultured on growth factor-reduced-matrigel were treated with BSA (1.3 nmol/L), Slit3 (0.80 nmol/L), VEGF (1.5 nmol/L), or FGF-2 (1.8 nmol/L). The images were acquired 6 hours after the treatments. (B). The total tube lengths of panel A were quantified. The data are summarized from experiments performed 3 times in triplicate and are presented as mean ± SD. *P < .05 compared with BSA control by Student t test. (C-D) The Anti-Robo4 Ab but not anti-Robo1 Ab inhibited Slit3-induced EC tube formation. Slit3-induced HUVEC tube formation was determined as described in panel A in the presence of 2 μg of nonimmune (Ni-Ab), anti-Robo1 (R1-Ab), or anti-Robo4 (R4-Ab) antibody (Ab). (E) Rat aortic ring assay. Rat aortic rings implanted in Matrigel supplemented with BSA (1.3 nmol/L), Slit3 (0.80 nmol/L), VEGF (1.5 nmol/L), or FGF-2 (1.8 nmol/L). The images were acquired 7 days after the treatments. Arrow indicates sprouting neomicrovessels. Arrowhead indicates migrating fibroblast cells. (F) Quantification of the sprouting neovessel followed the criteria described in “Methods.” The data are shown as an average score of each treatment ± SD, n = 6 for each test group. **P < .001 compared with BSA control by Wilcoxon signed-rank test. (G-H) The soluble extracellular domain of Robo4 but not of Robo1 inhibited Slit3-induced neovascularization in the rat aortic ring assays. Slit3-induced neovascularization was repeated with the rat aortic ring assay in the presence of BSA, soluble Robo1 (sR1), or soluble Robo4 (sR4). BSA and FGF-2 alone served as negative and positive control, respectively. **P < .001 compared with Slit3 + BSA group by Wilcoxon signed-rank test. n = 5 per test group.