March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
In Vitro And In Vivo Response Of VEGFR-2 To VEGF165 With/Without Ranibizumab
Author Affiliations & Notes
  • Amber Woolfenden
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Elizabeth Fassbender
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Siyuan Shen
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Kathyrn McAllister
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Lisa Baker
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Bruce D. Jaffee
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Stephen H. Poor
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Yubin Qiu
    Ophthamology Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts
  • Footnotes
    Commercial Relationships  Amber Woolfenden, None; Elizabeth Fassbender, None; Siyuan Shen, None; Kathyrn McAllister, None; Lisa Baker, None; Bruce D. Jaffee, None; Stephen H. Poor, None; Yubin Qiu, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2962. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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