June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
VEGF-Mediated Activation of Intracellular Signaling in Human Retinal Endothelial Cells is a Very Sharp and Switch-like Dose Response
Author Affiliations & Notes
  • Kenneth P Mitton
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Wendelin Dailey
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Austen N Knapp
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
    OUWB School of Medicine, Oakland University, Rochester, Michigan, United States
  • Peter F Chen
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
    OUWB School of Medicine, Oakland University, Rochester, Michigan, United States
  • Regan Miller
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Mei Cheng
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Brandon Metcalf
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Quentin Tompkins
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Ed Guzman
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Jennifer Felisky
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Mrinalini Deshpande
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Footnotes
    Commercial Relationships   Kenneth Mitton, None; Wendelin Dailey, None; Austen Knapp, None; Peter Chen, None; Regan Miller, None; Mei Cheng, None; Brandon Metcalf, None; Quentin Tompkins, None; Ed Guzman, None; Jennifer Felisky, None; Mrinalini Deshpande, None
  • Footnotes
    Support  NIH Grant: EY025089 (Mitton). VRRF: (Mitton)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1898. doi:
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      Kenneth P Mitton, Wendelin Dailey, Austen N Knapp, Peter F Chen, Regan Miller, Mei Cheng, Brandon Metcalf, Quentin Tompkins, Ed Guzman, Jennifer Felisky, Mrinalini Deshpande; VEGF-Mediated Activation of Intracellular Signaling in Human Retinal Endothelial Cells is a Very Sharp and Switch-like Dose Response. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1898.

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

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Abstract

Purpose : Elevated VEGFA (Vascular Endothelial Growth Factor-A) is a driver of neo-vascular and inflammatory response in diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Intraocular treatments to block VEGFA are effective but long-term consequences are of concern. Is it possible to titrate VEGF blockade? To elucidate the the activation kinetics of VEGFA on intracellular signaling within Human Retinal Endothelial Cells (HRECs), we developed an in situ assay to characterize the dose response for the VEGFA-mediated activation of the MAPK and AKT pathways.

Methods : Primary HRECS were cultured using endothelial specific media and techniques. For replicate dose response assays, the cells were seeded into a 96 well format. Complete monolayers were treated with human VEGFA-165. Post-VEGF treatment, HRECs were fixed and the active forms of MAPK and AKT were labeled in situ with primary antibodies and IR-fluorescent-tagged secondary antibodies. Beta-Actin protein was co-labeled in similar fashion. Fuorescenlt intensities were measured by IR-laser scanning, and the iactivated kinase values were normalized to beta-Actin. Dose response curves were fit using the R-project statistical package in Mac OS-X.

Results : The activation of both the MAPK and AKT pathways revealed that the dose response curves for VEGFA-165 have similar ED50 (effective dose, 50% activation) values in the 50-75 pM range. The dose response curves for both pathways revealed a sharp, pseudo-binary, activation response. A relatively small increase in VEGFA-165 concentration shifted the activation response of both pathways from baseline to maximum.

Conclusions : Our analysis revealed that VEGFA-165’s activation of intracellular signaling in primary HRECs has an extremely sharp dose response. A small increase in VEGFA concentration shifts intracellular pathways from a baseline activation state to maximum activation. This indicates that the MAPK and AKT activation response to VEGFA in Human Retinal Endothelial Cells is almost an on/off, switch-like, behavior. We conclude that it may be difficult to therapeutically achieve intermediate activation of intracellular signaling pathways in HRECs in vivo using current VEGF-blocking or trapping drugs.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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