FIGURE 6.

α_Mβ₂ supports angiogenesis via regulation of VEGF secretion by PMNs. (A) Peripheral blood WT, α_M^−/− or α_L^−/− PMNs (3×10⁶ cells/well) were incubated in 24-well TC plates in the absence or presence of TNFα (20ng/ml) for 2h at 37°C. Cycloheximide (10μg/ml) or pentoxifylline (300 μM) were added 60 min before addition of TNFα. VEGF concentration was measured in supernatants using mouse VEGF Quantikine Elisa Kit. Data are means ± SEM of triplicate samples and are representative of three independent experiments. (B) Bright field microscopy of tube formation by WT MAECs in the presence of conditioned media collected from WT, α_M^−/− or α_L^−/− peripheral blood PMNs stimulated with TNFα (upper panels). Inhibitors of VEGF: neutralizing anti-VEGF mAb, isotype matched rat IgG_2a (100 μg/ml) and recombinant mouse sFLT-1 (100ng/ml) were preincubated for 60 min with conditioned media of WT TNFα-stimulated PMNs before its addition to MAECs (lower panels). The images were taken after 6h incubation in 37°C, 5% CO₂. Scale bars, 75 μm. (C) Quantification of tube formation. Number of closed tubes was counted in 20 different fields of each treatment and plotted as mean ± SEM and are representative of two independent experiments.