Department of Ophthalmology, Justus Liebig University Giessen, Giessen, Germany.
Department of Ophthalmology, Justus Liebig University Giessen, Giessen, Germany; Department of Ophthalmology, Medical University of Innsbruck, Innsbruck, Austria.
Exp Eye Res. 2024 Oct;247:110062. doi: 10.1016/j.exer.2024.110062. Epub 2024 Aug 24.
Exposure to VEGF-Aa over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-Aa and this was more pronounced at lower concentrations of VEGF-Aa. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-Aa in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.
暴露于 VEGF-Aa 数天会导致牛视网膜的永生化内皮细胞（iBREC）形成的非常紧密的屏障持续功能障碍。屏障通透性升高的迹象包括通过细胞-基质阻抗测量确定的低细胞指数值、claudin-1 含量降低以及血管内皮钙黏蛋白在质膜上的均匀连续染色中断。由于发现血管生成生长因子血管生成素-2 可以调节 VEGF-A 对内在血视网膜屏障的有害影响，我们在体外更详细地研究了这种生长因子是否确实会改变视网膜内皮细胞形成的屏障的稳定性，或者调节 VEGF-A 的作用。鉴于抗 VEGF 治疗的临床相关性，我们还研究了阻断 VEGF-A 驱动的信号传导是否足以防止这两种生长因子组合引起的屏障功能障碍。尽管血管生成素-2 刺激 iBREC 的增殖，但在 3 nM 的浓度下形成的屏障不会减弱：细胞指数值仍然很高，claudin-1 和血管内皮钙黏蛋白的表达或亚细胞定位分别不受影响。血管生成素-2 增强了 VEGF-Aa 诱导的变化，并且在较低浓度的 VEGF-Aa 下更为明显。用 tivozanib 特异性抑制 VEGF 受体以及用 bevacizumab 干扰 VEGF-A 与其受体的结合可防止生长因子的有害作用；faricimab 对血管生成素-2 和 VEGF-A 的双重结合效率略高。通过添加 faricimab 可以有效地防止 iBREC 摄取细胞外血管生成素-2，该细胞也被该细胞内化。将细胞暴露于 faricimab 数天可稳定其屏障，证实抑制 VEGF-A 信号传导对这种细胞类型没有危害。总之，我们的结果证实了 VEGF-Aa 在导致视网膜内皮细胞通透性增加的过程中起主导作用，其中血管生成素-2 可能起次要调节作用。
