T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of quantification.
OBJECTIVE
A carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS) has demonstrated potent angiostatic activity during retinal developmental neovascularization in vivo. The effects of T2-TrpRS on pathologic neovascularization were tested and compared with a potent VEGF antagonist using the mouse model of oxygen-induced retinopathy (OIR).


METHODS
C57BL/6J mice were transiently exposed to hyperoxic conditions (75% O2) between postnatal day 7 (P7) and P12 and then returned to room air. Retinas were isolated, blood vessels stained with isolectin Griffonia simplicifolia, images of retinal whole-mounts acquired, and the area of vascular obliteration and extent of preretinal neovascularization quantified. This method was compared to the commonly used method of OIR quantification in which the number of pre-inner limiting membrane (ILM) nuclei is counted in serial sections of whole eyes. To assess the angiostatic activity of T2-TrpRS, mice were injected intravitreally at P12 with either T2-TrpRS, a VEGF aptamer, or vehicle (PBS) alone, and the effects on area of obliteration and on preretinal neovascular tuft formation were assessed.


RESULTS
Using a modified method of quantification in the mouse OIR model based on images of isolectin-stained retinal wholemounts, we were able to assess reliably and consistently both vascular obliteration and preretinal neovascular tuft formation in the same specimen. T2-TrpRS demonstrated potent angiostatic activity, reducing the appearance of pathologic neovascular tufts by up to 90%. Surprisingly, T2-TrpRS also enhanced physiological revascularization of the obliterated retinal vasculature, reducing these areas by up to 60% compared with PBS-injected eyes. In contrast, the VEGF antagonist, while similarly reducing preretinal neovascular tuft formation, did not enhance revascularization of the obliterated areas.


CONCLUSIONS
Use of a rapid, quantifiable method to assess the effect of T2-TrpRS on retinal angiogenesis in the OIR model demonstrates the importance of a quantification system that permits simultaneous analysis of a drug's effect on vascular obliteration as well as on preretinal neovascularization. The results obtained using this method suggest enhanced clinical value for compounds such as T2-TrpRS that not only inhibit pathologic neovascularization, but also facilitate physiological revascularization of ischemic tissue.
Banin
Eyal
E
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Dorrell
Michael I
MI
Aguilar
Edith
E
Ritter
Matthew R
MR
Aderman
Christopher M
CM
Smith
Alexandra C H
AC
Friedlander
Jeffrey
J
Friedlander
Martin
M
D043165:Angiostatic Proteins / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D000943:Antigens, Differentiation / Q000378:metabolism; D052158:Aptamers, Peptide / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D005451:Fluorescein Angiography; D019084:Fluorescent Antibody Technique, Indirect; D018496:Hyperoxia / Q000150:complications; D007267:Injections; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010100:Oxygen / Q000633:toxicity; D037121:Plant Lectins / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D015861:Retinal Neovascularization / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D012171:Retinal Vessels / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D014369:Tryptophan-tRNA Ligase / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D014822:Vitreous Body
D043165:Angiostatic Proteins; D000943:Antigens, Differentiation; D052158:Aptamers, Peptide; C045475:Griffonia simplicifolia lectins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; C036763:monocyte-macrophage differentiation antigen; C467480:vascular endothelial growth factor A, mouse; D014369:Tryptophan-tRNA Ligase; D010100:Oxygen