Endothelial monolayer permeability under controlled oxygen tension
Endothelial monolayer permeability under controlled oxygen tension
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DOI：
10.1039/c7ib00068e
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发表时间：
2017-06-01
影响因子：
2.5
通讯作者：
Kamm, Roger D.
中科院分区：
文献类型：
--
作者：
Funamoto, Kenichi;Yoshino, Daisuke;Kamm, Roger D.
内皮细胞的通透性在癌症等疾病的背景下得到了广泛的研究，也在血液循环的药物输送研究中得到了广泛的研究。缺氧是内皮细胞（EC）行为的重要调节因子，影响内皮细胞的屏障功能，但其作用研究甚少。本文利用微流控装置和普通细胞培养皿进行了细胞实验，揭示了缺氧对EC单层膜通透性的影响。将人脐静脉内皮细胞（HUVECs）植入一个微流控通道中，在中心腔室的胶原凝胶的每个垂直表面上形成EC单层。通过按预定比例将氧气、二氧化碳和氮气的气体混合物供应到装置中制造的通道中，调节氧张力以产生常氧（21% O-2）或缺氧（3% O-2）条件。通过分析荧光标记的葡聚糖在胶原凝胶中的扩散，定量的EC单层通透性随着缺氧暴露6小时后屏障功能丧失而增加，平均显示70 kDa和10 kDa葡聚糖分别增加11倍和4倍。与此一致的是，随后在培养皿上对HUVECs进行免疫荧光染色和单独的western blot分析显示，缺氧条件下ve -钙粘蛋白内化导致了松散的细胞-细胞粘附。因此，缺氧胁迫通过改变细胞-细胞连接的完整性来增加内皮细胞的通透性。
Endothelial permeability has been extensively investigated in the context of pathologies such as cancer and also in studies of drug delivery from the circulation. Hypoxia is a critical regulator of endothelial cell (EC) behavior and affects the barrier function of endothelial linings, yet its role has been little studied. This paper reveals the effect of hypoxia on the permeability of an EC monolayer by cellular experiments using a microfluidic device and a conventional cell culture dish. Human umbilical vein endothelial cells (HUVECs) were seeded into one microfluidic channel, creating an EC monolayer on each vertical surface of a collagen gel confined to a central chamber. Oxygen tension was regulated to produce normoxic (21% O-2) or hypoxic (3% O-2) conditions by the supply of gas mixtures of oxygen, carbon dioxide, and nitrogen at predefined ratios into channels fabricated into the device. Permeability of the EC monolayer quantified by analyzing diffusion of fluorescence-labelled dextrans into the collagen gel increases with barrier function loss by 6 hour hypoxic exposure, showing 11-fold and 4-fold increases for 70 kDa and 10 kDa dextrans, respectively, on average. Consistent with this, subsequent immunofluorescent staining and separate western blot analysis of HUVECs on a culture dish demonstrate loose cell-cell adhesion resulting from internalization of VE-cadherin under hypoxia. Thus, hypoxic stress increases endothelial permeability by altering cell-cell junction integrity.