Macrophages stimulated with C5a display an antiangiogenic phenotype. (A) Isolated primary bone marrow derived mouse macrophages treated with C5a showed a polarization toward an antiangiogenic phenotype as verified by significantly increased mRNA levels of IL-6, TNF-α, and soluble VEGFR-1 and significantly reduced levels of IL-10 mRNA using quantitative real-time PCR. IL-12 mRNA levels were not significantly changed by C5a treatment. The respective mRNA was normalized against actin. Data are mean ± SEM (n = 4) and are shown as percentage of control. mRNA of macrophages treated with vehicle control represents the 100% value. *P < .05. (B) Mouse macrophages were incubated with C5a (100nM) or vehicle control for 4 or 12 hours and supernatants were analyzed for sVEGFR1 by ELISA. Data are mean ± SEM (n = 3-4) and are shown as percentage of control. Detected sVEGFR1 in supernatant of control treated macrophages represents the 100% value. *P < .05. (C) HUVEC were treated with supernatants of human monocytes that were stimulated without (vehicle control) or with C5a (100nM) or C3a (100nM) for 12 hours and analyzed in the in vitro Matrigel tube formation assay. To assess for VEGF-induced tube formation, growth factor-reduced Matrigel supplemented with VEGF was used. The supernatant of C5a-stimulated human monocytes inhibited the VEGF induced tube formation of HUVEC. Length of forming tubes was quantified using Metamorph software. Data are mean ± SEM (n = 3) and are shown as percentage of control. Tube length of endothelial cells incubated with supernatant of control–treated macrophages represents the 100% value. *P < .05.