NFATc3 knock-down blocks COX-2-dependent in vivo angiogenesis. Matrigel plugs embedded with VEGF (500 ng/mL) and KDSc and KD-c3 human endothelial cells (2.5 × 105/mouse) were implanted in nude mice and 10 days later were collected and analyzed. (A) Hemoglobin was measured spectrophotometrically at 605 nm. Graph shows means ± SD from 3 independent experiments. (B) Matrigel plugs containing KDSc or KD-c3 HUVECs were paraffin embedded and sectioned. Vessel count is the number of vessels counted in H&E-stained sections (means ± SD; n = 3). (C) Paraffin sections of Matrigel plugs containing either KDSc or KD-c3 HUVECs were stained with anti-GFP antibody. Left panels are micrographs showing vessel-like structures formed by KDSc-expressing HUVECs (arrows). Right panels are KD-c3-expressing HUVECs unable to form vascular structures (black arrowheads); instead, vessels present in these plugs were mainly composed of GFP− host cells (white arrowheads). Bars indicate 100μm. (D) Micrographs showing vessels formed by GFP+ endothelium (left) with an evident lumen containing blood cells (asterisks). Bars indicate 50μm. *P < .05 and **P < .01 compared with control (KDSc).
