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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. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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: 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 wasassessed 1.) immunohistochemically on single cell level with a four-point semiquantitativegrading 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 westernblot (WB). Different incubation variables (temperature / time / BVC-concentration) werecompared. The amount of excreted BVC was measured in the cell culture medium.
Results: After 15 minutes incubation with 2.5 or 5 mg/ml BVC, 65±2% or 69±9% of all hTFshowed intracellular BVC (p=0.47 between groups). The semiquantitative grade of intracellularBVC staining (0=none, 3=100%) after 15 min for 2.5 and 5 mg/ml BVC was 1.03±0.35 and1.18±0.23, respectively (n=15; p=0,18). Longer incubation up to 90 min. did not significantlyincrease the uptake. At 4°C incubation the BVC uptake as measured with quantitative WB wassmaller at 15 min, but higher at 90 min. compared to 37°C incubation (p=0.003 and 0.01 for 2.5and 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 significantincrease of BVC in the medium after 60 min (1.6±1.7 vs 2.47±2; n=5, p=0.014) and a reductionof 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 mainlybound to cytoskeleton.
Conclusions: BVC is rapidly internalised into hTF. Endocytosis inhibiting conditions (4°C) slowdown but do not prevent the uptake. This suggests a non-endocytosis dependent uptake. Afternon-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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