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F. Altomare, S. Briggs, B. Graham, C. Smith, Z. Xu, S. R. Boyd; Systemic Delivery of PTK787, a VEGF- Receptor Tyrosine Kinase Inhibitor, Blocks Murine RetinoVascular Development. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1750.
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© ARVO (1962-2015); The Authors (2016-present)
To quantitatively determine inhibition of retinal angiogenesis using PTK787 in murine retinovascular development (RVD).
Mouse pups (n=4-21/dose) were treated with PTK787 succinate (Novartis Pharma AG, Switzerland) resuspended in PBS, injected sub-cutaneously once daily on PND0, 1, 2 and 3. Doses tested were 2.5, 10, 20, 25, 30 and 40mg/kg. Vehicle control animals received PBS. On PND4, pups were euthanised, and the eyes removed for Immunohistochemistry (IHC). Antibodies against Collagen IV (Chemicon), fluorescein-labeled secondary antibodies (Molecular Probes), and fluorescein-conjugated lectin (simplicifolia bandeira) were used to detect retinal vasculature. Processed retinas were photographed using a Nikon epifluorescent microscope at 40X magnification. Digital images of the total retinal surface were captured using an ACT1 image capture software (Nikon)and imported into Photoshop©, where a pre-built radial grid of eight equally spaced lines was overlaid and centered over the optic nerve. An Image Processing Tool Kit (Reindeer Graphics, Inc.) was used to measure the distance from the center of the optic nerve to the outermost blood vessel that each line crossed. The efficacy of PTK787 or vehicle was expressed as mean radial growth (+/- S.E.M) for each experimental group. Inter-group differences were analysed by one-way analysis of variance (ANOVA). Comparisons of experimental arms between several experiments (each with its own negative control) were analyzed by the repeated ANOVA. Pairwise comparisons of individual doses against control (in the final composite analyses) were analyzed using the Tukey t-test (in conjunction with one-way ANOVA). Note that the experiment was performed three times to ensure reproducibility. Animals were treated in accordance with ARVO and IACUC guidelines.
A total of 70 retinas were analyzed, and 513 radial measurements performed. The mean radial growth of the vehicle control vessel was 1015.1 +/- 32µm. For the PTK787 doses, radial growth was 983.4 +/- 43µm (2.5 mg/kg), 804.0 +/-57µm (10mg/kg), 269.8 +/-51µm (25mg/kg), 361.6 +/- 45µm (30mg/kg), and 221.8 +/-22µm (40mg/kg). This represents a growth reduction relative to vehicle of 3.1, 20.8, 73.4, 64.4, and 78.1%, respectively. The drug was well tolerated with equivalent losses in control and experimental groups.
Systemic administration of PTK787 blocked RVD in a dose-dependent manner, reaching an inhibition of 78% at maximum dose of 40 mg/kg.
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