Session Title: New Drug Treatment
Session Date/Time: Saturday 13/09/2014 | 14:30-16:30
Paper Time: 15:18
Venue: Boulevard F
First Author: : S.Hassan M. Aboul Naga EGYPT
Co Author(s): : M. Dithmer M. M. Soliman T. A. Macky J. Roider A. Klettner
Bevacizumab is taken up into RPE cells and is found intracellularly for a minimum of seven days. In this study, we investigate bevacizumab uptake and its intracellular localization at different time intervals in RPE cells.
The study was carried out at the Department of Ophthalmology of the University of Kiel, University Medical Center (Germany), in cooperation with the Ophthalmology Department of the Kasr Al Aini Cairo University Hospital (Egypt).
For this study, RPE cell line Arpe19 and primary porcine RPE cells, passage 2, were used. Cells were treated once with bevacizumab and intracellular bevacizumab was investigated after various time periods (1 h – 7 d). For the detection of internalized and RPE-bound bevacizumab, Western blot and immunofluorescence studies were utilized. For intracellular localization, antibodies against Clathrin, Rab5 (early endosome), Rab7 (late endosome), Lamp2 (lysosome), Myo7a (myosin motor) and CD63 (exosome) were used. Actin was stained using Phalloidin and Cytochalasin-D was administered to disrupt actin polymerization. Occludin was used to detail the cell membrane via its tight junctions, while WGA-Alexa Fluor® 647 was used to visualize the cell membrane itself.
Bevacizumab is abundantly found in porcine RPE cells and Arpe19 cells as detected in immunofluorescence and Western blot. After 1 h and 4 h of stimulation, bevacizumab is primarily located close to the cell membrane and can partly be found in close proximity to or colocalizing with Rab 5, indicating that sections of bevacizumab are taken up into early endosomes. First results indicate a possible involvement of Clathrin-coated pits in the uptake process. During 1 d to 4 days after bevacizumab challenge, intracellular bevacizumab displays a net-like pattern. After 7 d of bevacizumab challenge, the pattern of intracellular bevacizumab becomes more diffuse. Furthermore, early results indicate colocalization in the exosomal compartment. Bevacizumab is found in close proximity to or colocalizing with actin filaments, suggesting an actin-mediated intracellular transport that seems to be facilitated by myosin motor 7a. Colocalization with Lamp2 is rarely found, suggesting that bevacizumab is not degraded in lysosomes.
Bevacizumab displays a distinct, time dependent localization in RPE cells. The pattern suggests a controlled intracellular transport via actin filaments. Internalized bevacizumab does not seem to be transported into the lysosomal pathway and does not seem to be intracellularly degraded.