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Amber Woolfenden, Elizabeth Fassbender, Siyuan Shen, Kathyrn McAllister, Lisa Baker, Bruce D. Jaffee, Stephen H. Poor, Yubin Qiu; In Vitro And In Vivo Response Of VEGFR-2 To VEGF165 With/Without Ranibizumab. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2962.
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Establish and validate an in vitro and in vivo pharmacodynamic assay for VEGFR-2.
Human Umbilical Vein Embryonic Cells (HUVECs) were seeded in 96 well plates at 1X106 and allowed to adhere for 2 hours. Recombinant Human VEGF165 (rhVEGF165) in saline was added to cells at a concentration range of 0.025ng/ml to 1ug/ml and incubated at 37°C for 10 minutes. Plates were placed on ice, medium aspirated and cell lysis buffer added for 1 hour. Cell lysates were centrifuged and supernatant analyzed by enzyme-linked immunosorbent assay (ELISA) detecting VEGFR-2 autophosphorylation (p-VEGFR-2).Dutch Belted rabbits (n = 3 or 4 rabbits/group) were challenged with an intravitreal injection of 5, 10 or 20 µg rhVEGF165 in saline at a volume of 50 µl to both eyes. Rabbit eyes were harvested on ice 20 minutes post rhVEGF challenge, retina and retinal pigment epithelium (RPE)/choroid isolated and snap frozen. Tissues were later homogenized in the presence of protease and phosphatase inhibitors and retinal p-VEGFR-2 levels were analyzed by ELISA. 500 µg of Ranibizumab or vehicle control in a volume of 50 µl were injected into the vitreous of both eyes of rabbits (n = 3 or 4 rabbits/group). Rabbit eyes were then challenged with an intravitreal injection of 10 µg of rhVEGF165 in saline at a volume of 50 µl in one study 3 days after injection and in a second study at 5 days after injection of ranibizumab. Rabbit eyes were harvested after 20 minutes and retinal and RPE/choroid p-VEGFR-2 levels were analyzed by ELISA as described above.
Addition of rhVEGF165 to HUVEC cells demonstrated a dose dependant increase in levels of activated phospho-VEGFR-2 (rhVEGF EC50=62ng/ml). Intravitreal injection of rhVEGF165 at 5, 10, and 20 µg/eye resulted in approximately a 2 fold increase of retinal p-VEGFR-2 compared with saline injected controls, a dose response was not observed. Autophosphorylation of VEGFR-2 in the RPE/choroid was minimal under this experimental condition. Intravitreal ranibizumab injected at a dose of 500 µg/eye on day 3 or day 5 prior to 10 µg rhVEGF intravitreal challenge reduced p-VEGFR-2 in the retina by an average of 57% as compared to saline controls.
This model system may be used to assess pharmacodynamics effects of anti-VEGF agents and VEGFR-2 inhibitors.
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