Aboul Naga Shereen Hassan, Dithmer Michaela, Chitadze Guranda, Kabelitz Dieter, Lucius Ralph, Roider Johann, Klettner Alexa
University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany; University of Cairo, Kasr Al-Aini Faculty of Medicine, Cairo, Egypt.
University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
Exp Eye Res. 2015 Feb;131:29-41. doi: 10.1016/j.exer.2014.12.010. Epub 2014 Dec 19.
The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes.
抗血管内皮生长因子（VEGF）抗体贝伐单抗被广泛用于各种眼科疾病的非适应证治疗，最常见于年龄相关性黄斑变性和糖尿病性黄斑水肿。贝伐单抗能够穿透视网膜，并在玻璃体内注射后以时间依赖性方式在脉络膜中被发现。此前已证明它可被视网膜色素上皮（RPE）摄取。在本研究中，我们在原代猪RPE细胞和人细胞系ARPE19中都进行了研究，以探究贝伐单抗被RPE细胞摄取后的细胞内途径。通过免疫荧光和脉冲追踪实验检测发现，贝伐单抗呈现出一种特征性的、时间依赖性的细胞内分布模式。在原代细胞和细胞系中，通过免疫荧光和蛋白质印迹法检测发现，7天后细胞内可检测到贝伐单抗。应用后立即发现，贝伐单抗部分与Rab5共定位，表明在早期内体中有一定摄取。通过亚细胞分级分离和免疫荧光显示，在细胞内，贝伐单抗在细胞骨架部分被检测到，与肌动蛋白丝排列一致。通过三重染色免疫荧光测定，贝伐单抗似乎通过肌球蛋白7a沿着肌动蛋白丝移动。有趣的是，通过免疫荧光观察发现，在7天的时间里，贝伐单抗似乎在某些储存区域积累。此外，免疫细胞化学、蛋白质印迹法和流式细胞术获得的结果表明，贝伐单抗可能通过外泌体从RPE细胞中释放出来。总之，贝伐单抗以一种特征性的、时间依赖性的方式被视网膜色素上皮细胞摄取并在其中运输，它似乎通过肌球蛋白7a沿着肌动蛋白丝移动，并且似乎部分通过外泌体从细胞中释放出来。
