Schematic representation of human TyrRS and TrpRS constructs used in this study. Shaded regions of full-length TyrRS and TrpRS represent COOH- and NH₂-terminal appended domains, respectively. Numbers on the left and right correspond to the NH₂- and COOH-terminal residues relative to the human full-length enzymes, respectively. The COOH domain of TyrRS has sequence similarity to mature endothelial monocyte-activating polypeptide II (45). The NH₂ domain of TrpRS has sequence similarity to the extra domains of human GluProRS (a fusion of glutamyl- and prolyl-tRNA synthetases), MetRS, GlyRS, and HisRS (8, 10–15).

Activities of human TrpRS constructs on HUVECs. (A) Inhibition of HUVEC migration by human TrpRS constructs. Average number of cells per high-power field migrating in response to stimulation by cell medium, VEGF (0.5 nM), VEGF (0.5 nM) + full-length TrpRS (500 nM), VEGF (0.5 nM) + mini TrpRS (500 nM). (B) Inhibition of endothelial cell proliferation by human TrpRS constructs. Human TrpRS (2 μM) were assayed on HUVEC cells in the presence of VEGF₁₆₅ (2 nM) in a 72-h proliferation experiment. Cell proliferation is presented as the percentage of net VEGF₁₆₅-induced (2 nM) proliferation. Values represent the mean ± standard deviation from five experiments.

Proteolytic cleavage of human TrpRS. A protease:protein ratio of 1:3,000 was used. The cleavage was done at 37°C in PBS (pH 7.4) for 0 min (lane 1), 15 min (lane 2), 30 min (lane 3), or 60 min (lane 4). Samples were analyzed on SDS/12.5% polyacrylamide gels. Molecular size markers are given to the left (in kilodaltons).

Activity of human TrpRS in the chicken chorioallantoic membrane assay. The data are presented as the mean number of blood vessel branches ± standard deviations from 5–8 embryos in each sample. (A) Angiostatic activity of human TrpRS fragments. PBS, VEGF₁₆₅ (1 pmol), VEGF₁₆₅ (1 pmol) + IP10 (120 pmol), VEGF₁₆₅ (1 pmol) + full-length TrpRS (60 pmol), VEGF₁₆₅ (1 pmol) + mini TrpRS (60 pmol), or VEGF₁₆₅ (1 pmol) + T1-TrpRS (60 pmol). VEGF₁₆₅ was added on day 0; IP10 and TrpRS were added for three consecutive days starting at day 1. (B) Digital images of representative filter discs showing angiogenesis (CAM assays) stimulated by VEGF₁₆₅ and inhibited by mini TrpRS.

Activity of human TrpRS in a murine matrigel model of angiogenesis. Athymic wehi mice were s.c. implanted with 400 μl of growth factor-depleted matrigel containing an angiogenic stimulus. Intraperitoneal injections (i.p.) of mini TrpRS were given on days 2, 3, and 4. On day 5, the mice were intravenously injected with the fluorescein-labeled endothelial binding lectin Griffonia (Bandeiraea) Simplicifolia I, isolectin B4. The plugs were resected and solubilized, and the fluorescein content was quantitated by spectrometry. (A) Relative fluorescein content of resected matrigel plugs treated with medium, VEGF₁₆₅ (8 pmol), VEGF (8 pmol) + i.p. injected mini TrpRS (1 nmol), VEGF₁₆₅ (8 pmol) + mini TrpRS (1 nmol, included in matrigel). (B) Fluorescent images of resected matrigel plugs by using confocal microscopy. Stimulation of angiogenesis by VEGF₁₆₅ and inhibition of VEGF₁₆₅-stimulated angiogenesis by mini TrpRS. In the absence of VEGF, the images are similar to those shown on the right.

Fragments of TrpRS inhibit angiogenesis in a postnatal mouse retinal model. Representative images of superficial (primary layer) and deep (secondary layer) retinal vasculature observed after intravitreal injection of mini TrpRS (Upper) or full-length TrpRS (Lower). On postnatal day 8, eyes were treated by intravitreous injection of PBS, human full-length TrpRS (5 pmol), or mini TrpRS (5 pmol), and retinas were harvested on postnatal day 12 or 13. Blood vessels were visualized after staining with anti-collagen IV antibodies. Qualitative image analysis of retinal vessel formation between P8 and P12 showed complete inhibition of the secondary layer in 28% of eyes treated with mini TrpRS. Full-length TrpRS had no effect on secondary layer formation. Note that the primary layer was unaffected by either mini or full-length TrpRS.