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First published online 15 June 2005
doi: 10.1242/dev.01888

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Angiopoietin 1 causes vessel enlargement, without angiogenic sprouting, during a critical developmental period

¹ Regeneron Pharmaceuticals Incorporated, 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA
² Cardiovascular Research Institute, UCSF Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA

View larger version (108K): [in a new window]   Fig. 1. Reddened skin in mouse and rat pups treated with angiopoietin 1. (A-C) Snouts of wild-type adult mice (A) are pale compared with the reddened snout of the transgenic K14-ANG1 mouse (B), which overexpresses angiopoietin 1 in the skin. Prolonged treatment (10 days) of normal adult mice with ANG14FD protein (C) or Adeno-ANG1* adenovirus (not shown) does not cause reddening. (D) Snouts of mouse pups treated for 7 days (beginning at P7) with PBS or ANG14FD (200 µg ip). The snout (arrow), gums and tongue of the pups treated with ANG14FD are markedly reddened compared with the PBS-treated pup. (E,F) Snouts and ears of rat pups treated for 7 days (beginning at P7) with PBS or ANG14FD. The snouts (arrow), gums, tongues, paws (arrow) and ears (arrow) of rat pups treated with ANG14FD (200 µg ip) are markedly reddened compared with PBS-treated pups.

View larger version (110K): [in a new window]   Fig. 2. Vessel morphology in mouse tissues after treatment with angiopoietin 1. Mouse pups were treated daily for 7 days with 200 µg of ANG14FD protein. Arteries and veins were readily identified in wholemounts by virtue of the differential coating of the smooth muscle cell layer (arterioles had a regular layer of elongated smooth muscle cells wrapped circumferentially around the vessels, whereas venules had an irregular layer of spread, branched smooth muscle cells associated with the vessels) and by vessel morphology (arteries were straighter and smaller in diameter than the corresponding veins). (A,B) Whole-mount views of tracheas from P14 mice, with blood vessels immunostained for PECAM (green) and -smooth muscle cell actin (red/orange). The straight capillaries traverse the cartilaginous rings. The venular ends of the capillaries are enlarged in the tracheas of ANG14FD-treated mice (arrows, B) compared with PBS controls (A), and the draining venules are also enlarged, whereas the arterioles (arrowheads) are not enlarged. (C,D) Cross-sections of tongue from P14 mice, with blood vessels immunostained for PECAM (green) and -smooth muscle cell actin (red/orange). The upper epidermal surface of the tongue is at the upper part of the image. The draining venules and the vessel loops in the dermal papillae (arrows) in the muscosa and muscle layers are enlarged in the tongues of ANG14FD-treated mice (D) compared with PBS controls (C). By contrast, the feeding arterioles (arrowheads) are similar in size in both ANG14FD-treated and control mice. (E,F) Whole-mount views of diaphragm from P14 mice, with blood vessels immunostained for PECAM (green) and -smooth muscle cell actin (red/orange). The straight capillaries are in the skeletal muscle and the draining venules (arrows) are at the boundary with the central tendon. The venules (arrows) in the central tendon are enlarged in the diaphragms of ANG14FD-treated mice (F) compared with PBS controls (E). By contrast, the muscle capillaries in ANG14FD-treated mice and control mice are similar in size. (G,H) Whole-mount views of retinas from P14 mice, with blood vessels stained with Griffonia simplicifolia isolectin B4 (green). The capillaries traverse between arterioles (arrowheads) and venules (arrows). The venular networks (arrows) are enlarged in retinas of ANG14FD-treated mice (H) compared with PBS controls (G).

View larger version (91K): [in a new window]   Fig. 3. (A,B) Time course and dose-response analysis of vessel enlargement after treatment with ANG14FD. 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 ANG14FD, 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 ANG14FD. Results are mean±s.e.m.; three mice per group. (C) TIE2 phosphorylation after treatment with ANG14FD, and inhibition by ANG12FD. 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, ANG12FD, ANG14FD, or ANG12FD plus ANG14FD. TIE2 from lung shows a basal level of tyrosine phosphorylation in PBS-treated mice, which was not significantly altered by treatment with ANG12FD. ANG14FD induced increased TIE2 phosphorylation, which was largely inhibited by ANG12FD. 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 ANG12FD, ANG14FD, 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 ANG12FD (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 ANG14FD treatment (F) was largely inhibited by co-treatment with ANG12FD (G), whereas arterioles (arrows) were not affected by ANG14FD. (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 ANG12FD (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 ANG14FD treatment (I) was largely inhibited by co-treatment with ANG12FD (J), whereas arterioles (arrows) were not affected by ANG14FD.

View larger version (92K): [in a new window]   Fig. 4. Vessel enlargement is associated with increased numbers of endothelial cells and endothelial cell proliferation. Retinal vessels in wholemounts of PBS- or ANG14FD-treated mice, stained with BrdU and Cy3-labeled secondary antibody. (A) In control mice, BrdU label (red) is apparent near the retinal periphery where active angiogenesis occurs in P14 mice, as well as in veins (arrows). (B) In ANG14FD-treated mice, intense BrdU labeling is apparent in veins (arrows) and in microvessels near veins. (C) Higher magnification of retinal vessels (labeled with isolectin B4, green) shows occasional BrdU-labeled (red) endothelial cells in veins (arrow), and few BrdU cells in arterioles (arrowhead) in control mice. (D) By contrast, in retinal vessels of mice treated with ANG14FD, BrdU-labeled endothelial cells are abundant in venous capillaries and veins (arrows). (E) Quantification of BrdU labeling shows approximately four times as many BrdU-labeled endothelial cells in retinal vessels from mice treated with ANG14FD as in PBS-treated mice.

View larger version (117K): [in a new window]   Fig. 5. Most vessels lose responsiveness to ANG14FD between P14 and P30. Mice were injected daily for 7 days starting at P7, P14, P30 and P49 with ANG14FD. Vessels were immunostained for PECAM (green) and -smooth muscle cell actin (red). (A,B) Snouts of mice treated with ANG14FD (arrows) were dramatically reddened in mice treated at P14 (A), but not at P30 (B). (C-E) Enlarged tracheal vessels in ANG14FD-treated P14 (D) and P30 (E) mice compared with control mice (C). Enlargement occurred in venular capillaries, in postcapillaries (arrows) and in collecting venules, whereas arterioles (arrowheads) appeared to be unchanged. (F-H) Vessels in the tongue of P14 ANG14FD-treated mice (G) are enlarged compared with controls (F), but those from P30 mice (H) are only slightly enlarged. Enlargement was apparent in vessels of dermal papillae and in draining venules (arrows), whereas arterioles (arrowheads) appear to be unchanged. (I,J) Tracheal vessels of adult mice (12 wk) at 14 days after intranasal administration of adenovirus encoding green fluorescent protein (I) or angiopoietin 1* (J). Angiopoietin 1* caused enlargement of airway venules (arrows).

View larger version (163K): [in a new window]   Fig. 6. Vessel morphology in mouse tissues treated with VEGF-Trap. Mouse pups were treated every 2 days for 7 days with 25 mg/kg ip of VEGF-Trap protein. Tissue is from P14 mice, with blood vessels immunostained for PECAM (green) and smooth muscle cell actin (red/orange). (A,B) Whole-mount views of tracheas, showing straight vessels (arrowheads) across the cartilaginous rings in control mice (A). The vessels across the cartilaginous region (asterisks) in tracheas of VEGF-Trap-treated mice (B) are completely absent. (C,D) Cross-sections of tongue, with the upper epidermal surface of the tongue at the upper part of the image. The vessel loops in the dermal papillae (C, arrowheads) in tongues of VEGF-Trap-treated mice are almost abolished (D, asterisks), and the vascularity of the dermis and muscle layers is also reduced.