Department of Ophthalmology, Ulm University Medical Center, Ulm, Germany.
Department of Ophthalmology, Justus-Liebig-University Giessen, Friedrichstrasse 18, 35392, Giessen, Germany.
Sci Rep. 2022 Jul 21;12(1):12493. doi: 10.1038/s41598-022-16770-7.
Because rare, but severe adverse effects, i.e. retinal vasculitis or retinal vein occlusion, have been observed after repetitive intravitreal injections of VEGF-A-binding single-chain variable fragment brolucizumab (Beovu), we investigated its possible impact on the barrier formed by immortalized bovine retinal endothelial cells (iBREC) in comparison to that of the VEGF-A-binding Fab fragment ranibizumab (Lucentis). As a measure of stability of the barrier formed by a confluent monolayer of iBREC, we determined the cell index over seven days by continuous electric cell-substrate impedance measurements: Beovu but not Lucentis indeed significantly lowered the cell index, evident about 1.5 days after its addition, pointing to barrier impairment. Early after addition of Beovu, amounts of the integrins α5 and β1-subunits of the fibronectin receptor-had changed in opposite ways, suggesting an effect on cell adhesion due to hindered dimer formation. After exposure for eight days to Beovu, levels of claudin-1-an essential part of the iBREC barrier-were significantly lower, less claudin-1 was located at the plasma membrane after exposure to the VEGF-A antagonist for five days. Beovu did not induce secretion of inflammatory cytokines or VEGF-A. Interestingly, polysorbate-80-component of Beovu-but not polysorbate-20-in Lucentis-slightly, but significantly lowered the cell index, also associated with reduced claudin-1 expression. In summary, our results indicate that Beovu changes the behavior of retinal endothelial cells, thus providing an alternative "non-immunological" explanation for the most relevant of observed side effects.
由于反复玻璃体内注射 VEGF-A 结合单链可变片段 brolucizumab（Beovu）后观察到罕见但严重的不良反应，如视网膜血管炎或视网膜静脉阻塞，我们研究了它对永生化牛视网膜内皮细胞（iBREC）形成的屏障的可能影响，与 VEGF-A 结合 Fab 片段 ranibizumab（Lucentis）进行比较。作为 iBREC 融合单层形成的屏障稳定性的衡量标准，我们通过连续的电细胞-底物阻抗测量在七天内确定细胞指数：Beovu 而不是 Lucentis 确实显著降低了细胞指数，在添加后约 1.5 天明显，表明屏障受损。Beovu 添加后早期，纤连蛋白受体的整合素 α5 和 β1 亚基的含量以相反的方式发生变化，表明由于二聚体形成受阻对细胞黏附产生影响。在暴露于 Beovu 八天后， Claudin-1 的水平 - iBREC 屏障的重要组成部分 - 明显降低，在暴露于 VEGF-A 拮抗剂五天后 Claudin-1 位于质膜的位置较少。Beovu 不会诱导炎症细胞因子或 VEGF-A 的分泌。有趣的是，Beovu 的聚山梨醇酯-80 成分 - 而不是 Lucentis 中的聚山梨醇酯-20 - 轻微但显著降低了细胞指数，也与 Claudin-1 表达减少有关。总之，我们的结果表明，Beovu 改变了视网膜内皮细胞的行为，从而为观察到的副作用中最相关的副作用提供了一种替代的“非免疫”解释。
