A pulse of TNF promotes angiogenesis in vitro. (A) EC-coated beads in fibrin gels were treated with TNF (10 ng/mL) or PBS control and photographed after 4 days on an Olympus IX70 inverted phase-contrast microscope using a 4×/0.13 NA objective and an Optronics digital camera. High-resolution images were magnified in Photoshop. Control cultures showed robust sprouting, whereas TNF almost completely blocked sprouting. One of multiple similar experiments. (B) Cultures were established as in panel A and TNF was added at day 0, 2, or 4. The number of sprouts was counted at day 6. Shown are means and SD; *P < .005 relative to control, by Student t test. One of 2 similar experiments. (C) Cultures were established as above and treated for 2 days with or without TNF (10 ng/mL). TNF-containing medium was then removed and fresh medium lacking TNF was added every 2 days. Sprouts were counted at the indicated times. Shown are means and SD; *P < .005 relative to control, by Student t test. One of 3 similar experiments. (D) ECs (4 × 10⁴) were plated in 24-well plates in the presence or absence of TNF (10 ng/mL) and counted at the indicated times. *P < .005 relative to control, by Student t test. One of 2 similar experiments. (E) TNF blocks VEGF signaling acutely. ECs were pretreated with TNF (10 ng/mL) for 2 days, rested for 1 day, and then given VEGF (10 ng/mL) for 4 hours (TNF then VEGF), or were given TNF for 3 days and then TNF + VEGF for the last 4 hours (TNF + VEGF), or were given VEGF alone for 4 hours (VEGF) or left untreated (control). RNA was harvested for analysis of α2-macroglobulin expression by qRT-PCR. Supernatants were also collected from an independent experiment for analysis of α2-macroglobulin protein level by enzyme-linked immunosorbent assay (ELISA) (inset).