July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
Intracellular uptake of Bevacizumab in human Tenon-Fibroblasts
Author Affiliations & Notes
  • Florian Hasan
    University Medical Center Göttingen, Göttingen, Germany
  • Christian van Oterendorp
    University Medical Center Göttingen, Göttingen, Germany
  • Charlotte Fischer
    University Medical Center Göttingen, Göttingen, Germany
  • Nicolas Feltgen
    University Medical Center Göttingen, Göttingen, Germany
  • Hans Hoerauf
    University Medical Center Göttingen, Göttingen, Germany
  • Footnotes
    Commercial Relationships   Florian Hasan, None; Christian van Oterendorp, None; Charlotte Fischer, None; Nicolas Feltgen, None; Hans Hoerauf, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4726. doi:
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    • Get Citation

      Florian Hasan, Christian van Oterendorp, Charlotte Fischer, Nicolas Feltgen, Hans Hoerauf; Intracellular uptake of Bevacizumab in human Tenon-Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4726.

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

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Abstract

Purpose : Purpose: The use of the Vascular Endothelial Growth Factor (VEGF)-inhibitor Bevacizumab
(BVC) is a new approach of inhibiting fibrotic wound healing after glaucoma filtration surgery.
BVC is internalised by human tenon fibroblasts (hTF), but little detail is known about this uptake.
We investigated the dynamics of uptake and the intracellular distribution of BVC.

Methods : Methods: Primary hTF were incubated with BVC (2.5 mg/ml (non-lethal) and 5 mg/ml (lethal >
6h)) at 37°C, and, to inhibit endosomal uptake, also at 4°C. Intracellular uptake of BVC was
assessed 1.) immunohistochemically on single cell level with a four-point semiquantitative
grading scale and 2.) using quantitative western blot for IgG. To identify intracellular BVC-
binding compartments, hTF cell lysate was fractionated and analysed for IgG-levels via western
blot (WB). Different incubation variables (temperature / time / BVC-concentration) were
compared. The amount of excreted BVC was measured in the cell culture medium.

Results : Results: After 15 minutes incubation with 2.5 or 5 mg/ml BVC, 65±2% or 69±9% of all hTF
showed intracellular BVC (p=0.47 between groups). The semiquantitative grade of intracellular
BVC staining (0=none, 3=100%) after 15 min for 2.5 and 5 mg/ml BVC was 1.03±0.35 and
1.18±0.23, respectively (n=15; p=0,18). Longer incubation up to 90 min. did not significantly
increase the uptake. At 4°C incubation the BVC uptake as measured with quantitative WB was
smaller at 15 min, but higher at 90 min. compared to 37°C incubation (p=0.003 and 0.01 for 2.5
and 5 mg/ml).
Pulsed incubation with 5 mg/ml BVC for 30 min. at 37°C did not induce significant cell death.
WB of cell lysate and culture medium at 60 and 360 min. after BVC pulse showed a significant
increase of BVC in the medium after 60 min (1.6±1.7 vs 2.47±2; n=5, p=0.014) and a reduction
of cellular BVC after 360 min (0.22±0.04 vs. 0.1±0.02 (GAPDH normalised); n=5, p=0.038).
In WB from fractioned cell lysate after 60 min. BVC incubation at 37° and 4°, IgG was mainly
bound to cytoskeleton.

Conclusions : Conclusions: BVC is rapidly internalised into hTF. Endocytosis inhibiting conditions (4°C) slow
down but do not prevent the uptake. This suggests a non-endocytosis dependent uptake. After
non-lethal BVC pulses, hTF were able to excrete BVC.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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