Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School.
Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School;
J Vis Exp. 2022 Jun 7(184). doi: 10.3791/64076.
Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.
血视网膜屏障 (BRB) 的功能障碍导致了几种血管性眼病的病理生理学改变，常导致视网膜水肿和随后的视力丧失。内血视网膜屏障 (iBRB) 主要由视网膜血管内皮细胞组成，在生理条件下具有低通透性。这种低通透性的特征是通过相邻的视网膜微血管内皮细胞之间的旁细胞转运率低以及通过它们的跨细胞转运（胞吞作用）来紧密调节和维持的。视网膜跨细胞屏障通透性的评估可为健康和疾病状态下 iBRB 完整性提供基本的见解。在这项研究中，我们描述了一种内皮细胞 (EC) 胞吞作用测定法，作为评估 iBRB 通透性的体外模型，使用人视网膜微血管内皮细胞 (HRMEC)。该测定法评估了 HRMEC 以受体和小窝介导的跨细胞转运过程分别运输转铁蛋白和辣根过氧化物酶 (HRP) 的能力。在多孔膜上培养的完全汇合的 HRMEC 用荧光标记的转铁蛋白（网格蛋白依赖性胞吞作用）或 HRP（小窝介导的胞吞作用）孵育，以测量转移到下室的转铁蛋白或 HRP 的水平，指示 EC 单层的胞吞作用水平。已知调节 iBRB 的 Wnt 信号被调节以证明基于 HRP 的小窝介导的胞吞作用测定方法。这里描述的 EC 胞吞作用测定法可能为研究血管性疾病中 EC 通透性和 iBRB 完整性的分子调节剂以及筛选药物输送系统提供有用的工具。
