Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability.
Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability.
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DOI：
10.3791/64076
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发表时间：
2022-06-07
期刊：
影响因子：
--
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Bora K;Wang Z;Yemanyi F;Maurya M;Blomfield AK;Tomita Y;Chen J
血视网膜屏障（BRB）的功能障碍有助于几种血管性眼病的病理生理，通常导致视网膜水肿和随后的视力丧失。生理条件下，血视网膜内屏障主要由低通透性的视网膜血管内皮构成。这种低通透性的特征是由邻近视网膜微血管内皮细胞之间的低细胞旁转运率以及通过它们的跨细胞转运（胞吞作用）严格调节和维持的。视网膜跨细胞屏障通透性的评估可能为iBRB在健康和疾病中的完整性提供基本的见解。在本研究中，我们使用人视网膜微血管内皮细胞（HRMECs），描述了内皮细胞（EC）胞溶试验，作为评估iBRB通透性的体外模型。该试验分别评估了hrmec在受体和小泡介导的跨细胞转运过程中转运转运蛋白和辣根过氧化物酶（HRP）的能力。在多孔膜上培养的完全融合的hrmec与荧光标记的转铁蛋白（网格蛋白依赖的胞吞作用）或HRP（小泡介导的胞吞作用）一起孵育，以测量转移到底腔的转铁蛋白或HRP的水平，表明EC单层的胞吞作用水平。Wnt信号是一种已知的调节iBRB的途径，该途径被调节，以证明基于酶解酶的小泡介导的胞吞实验方法。本文所述的EC细胞吞噬试验可以为研究血管病理中EC渗透性和iBRB完整性的分子调节因子和筛选药物输送系统提供有用的工具。
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.
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影响因子：
1.8
作者：
Díaz-Coránguez M;Ramos C;Antonetti DA
通讯作者：
Antonetti DA
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2.3
作者：
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影响因子：
64.8
作者：
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Silver, David L.
DOI：
10.1073/pnas.0808698106
发表时间：
2009-02-10
影响因子：
11.1
作者：
Argaw, Azeb Tadesse;Gurfein, Blake T.;John, Gareth R.
通讯作者：
John, Gareth R.
影响因子：
64.8
作者：
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通讯作者：
Gu, Chenghua