Fig. 3. (A,B) Time course and dose-response analysis of vessel enlargement after treatment with ANG1^4FD. The diameter of tracheal vessels on the edge of cartilaginous rings was measured in confocal micrographs of PECAM-stained tracheal wholemounts. (A) Time course of venule enlargement in mice treated daily with 200 µg/day of ANG1^4FD, starting at P7. Venules increased in diameter with treatment (black bars), whereas untreated venules (white bars) did not change in diameter. Arterioles and venules were identified as described for Fig. 2. (B) Dose-response analysis of vessel enlargement. Mice treated daily for 7 days (starting at P7) with 20, 50 or 200 µg/day of ANG1^4FD. Results are mean±s.e.m.; three mice per group. (C) TIE2 phosphorylation after treatment with ANG1^4FD, and inhibition by ANG1^2FD. Lungs of mice treated daily for 7 days starting at P7 were excised, extracted, immunoprecipitated for TIE2, and immunoblotted for phosphotyrosine (upper panel) or TIE2 (lower panel). Mice were treated with PBS, ANG1^2FD, ANG1^4FD, or ANG1^2FD plus ANG1^4FD. TIE2 from lung shows a basal level of tyrosine phosphorylation in PBS-treated mice, which was not significantly altered by treatment with ANG1^2FD. ANG1^4FD induced increased TIE2 phosphorylation, which was largely inhibited by ANG1^2FD. Results shown are from one mouse per group, but are representative of a total of six mice per group analyzed for TIE2 phosphorylation. (D) Reduced vessel diameter with inhibitor of angiopoietin 1. Diameter of tracheal vessels on the edge of cartilaginous rings was measured in confocal micrographs of PECAM-stained tracheal wholemounts. Mice treated daily for 7 days with 200 µg/day of ANG1^2FD, ANG1^4FD, or both, starting at P7. Results are mean±s.e.m.; three mice per group. (E-J) Reduced vessel enlargement with an inhibitor of angiopoietin 1. Blood vessels were immunostained for PECAM (green) and -smooth muscle cell actin (red/orange). (E-G) Whole-mount views of tracheas from P14 mice. Daily treatment of mice with ANG1^2FD (200 µg/day) for 7 days had no obvious effect on vessel morphology (E). The enlargement of the venules near the cartilaginous rings (arrows) induced by ANG1^4FD treatment (F) was largely inhibited by co-treatment with ANG1^2FD (G), whereas arterioles (arrows) were not affected by ANG1^4FD. (H-J) Cross-sections of tongue from P14 mice. The epithelial surface of the tongue is at the upper part of the image. Daily treatment of mice with ANG1^2FD (200 µg/day) for 7 days (H) had no obvious effect on vessel morphology. The enlargement of the vessels in the dermal papillae (arrowheads) and draining venules (arrows) induced by ANG1^4FD treatment (I) was largely inhibited by co-treatment with ANG1^2FD (J), whereas arterioles (arrows) were not affected by ANG1^4FD.