June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Title. Development of human retinal pigment epithelial cells with inducible VEGF expression
Author Affiliations & Notes
  • Inesa Lelyte
    Department of Ophthalmology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States
    Institute of Inflammation and Ageing, University of Birmingham, Birmingham, Birmingham, United Kingdom
  • Vidhya R. Rao
    Department of Ophthalmology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States
  • Zubair Ahmed
    Institute of Inflammation and Ageing, University of Birmingham, Birmingham, Birmingham, United Kingdom
  • Giedrius Kalesnykas
    R&D Division, Experimentica Ltd, Kuopio, Finland
    R&D Division, Experimentica Ltd, Vilnius, Lithuania
  • Simon Kaja
    Departments of Ophthalmology and Molecular Pharmacology & Neuroscience, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States
    R&D Division, Experimentica Ltd, Forest Park, Illinois, United States
  • Footnotes
    Commercial Relationships   Inesa Lelyte Experimentica Ltd, Code E (Employment); Vidhya Rao None; Zubair Ahmed None; Giedrius Kalesnykas Experimentica Ltd., Code E (Employment), Experimentica Ltd., Code F (Financial Support), Experimentica Ltd., Code I (Personal Financial Interest), Experimentica Ltd., Code R (Recipient), Experimentica Ltd., Code S (non-remunerative); Simon Kaja Experimentica Ltd., Code C (Consultant/Contractor), Experimentica Ltd., Code E (Employment), Experimentica Ltd., K&P Scientific LLC, Code F (Financial Support), Experimentica Ltd., K&P Scientific LLC, Code I (Personal Financial Interest), eyeNOS Inc., Code P (Patent), Experimentica Ltd., K&P Scientific LLC, Code R (Recipient), Experimentica Ltd., K&P Scientific LLC, Code S (non-remunerative)
  • Footnotes
    Support  European Commission Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network Project #813440, Ocular Research By Integrated Training And Learning (ORBITAL), Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology, Richard A. Perritt M.D. Charitable Foundation, Illinois Society for Prevention of Blindness, Experimentica Ltd.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3608 – A0063. doi:
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      Inesa Lelyte, Vidhya R. Rao, Zubair Ahmed, Giedrius Kalesnykas, Simon Kaja; Title. Development of human retinal pigment epithelial cells with inducible VEGF expression. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3608 – A0063.

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

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Abstract

Purpose : Vascular endothelial growth factor A (VEGF) is upregulated in neovascular ocular conditions, including diabetic retinopathy (DR). To study the pathological processes in DR and develop novel treatments, VEGF-induced animal models have been established. Intravitreal injections of recombinant VEGF have been shown to mimic many of the complex DR mechanisms. However, the effects are transient due to the short half-life of injected peptides. The aim of this study was to generate an inducible VEGF expression the human retinal pigment epithelium (ARPE-19) cells.

Methods : ARPE-19 cells were transduced with lentivirus (LV) expressing a SparQTM all-in-one cumate-inducible plasmid (System Biosciences) expressing VEGF-A165 at a multiplicity of infection (MOI) of 20. To induce VEGF expression, cumate (30 μg/ml) was added 3d post-transduction. After 48h, VEGF levels were determined in the culture medium by ELISA (R&D Systems) and by qPCR from cell lysates. Cell viability was evaluated by MTT assay; cell motility was measured by scratch assays. Effects of VEGF expression on ARPE19 permeability were performed in Transwell® inserts (Corning Inc.) using conditioned media and quantified using fluorescent permeability standard, 6-carboxyfluorescein (6-CF).

Results : Cumate induction resulted in a 72% increase in VEGF levels as assessed by ELISA (MOI 20: 2005 ± 270 pg/ml vs. Control: 1165 ± 192 pg/ml, p < 0.05). qPCR revealed a 2.5-fold increase of VEGF mRNA levels compared with control (p < 0.01). MTT assay showed significantly increased number of cells at MOI 20 (115 ± 5%) compared with Control (100 ± 1%, p < 0.05), suggesting increased rate of proliferation. Permeability across the RPE monolayer was significantly increased in LV-transduced cells. Treatment of ARPE19 cells with conditioned media resulted in a significant increase in the apparent permeability coefficients (Papp) for 6-CF (0.66 ± 0.08 vs. 1.80 ± 0.41 x 105 cm/s, p < 0.05). Scratch assays revealed increased motility and presence of tube-like structures in transduced cells, suggesting the presence of VEGF.

Conclusions : Cumate-inducible VEGF expression is a feasible approach for mechanistic studies evaluating the dose-dependent effects of VEGF on ARPE19 in preclinical drug discovery studies. Future work will generate stably expressing cell lines and evaluate the utility of cumate-inducible VEGF-expressing LV in in vivo systems.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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