In Vitro And In Vivo Response Of VEGFR-2 To VEGF165 With/Without Ranibizumab

Abstract

Purpose: : Establish and validate an in vitro and in vivo pharmacodynamic assay for VEGFR-2.

Methods: : 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.

Results: : 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.

Conclusions: : This model system may be used to assess pharmacodynamics effects of anti-VEGF agents and VEGFR-2 inhibitors.

Keywords: vascular endothelial growth factor • retina • phosphorylation