Department of Surgery, Yale University, New Haven, Connecticut 06520-8062, USA.
Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3216-25. doi: 10.1167/iovs.09-4162. Epub 2009 Dec 30.
Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood-retinal barrier were examined.
Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K(+) and Na(+) (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG.
Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K(+), but did not discriminate between Na(+) and Cl(-). VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K(+), Na(+), and Cl(-). They had minimal effects on the expression and distribution of the claudins.
RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K(+) over Na(+) and Cl(-). Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab.
贝伐单抗和雷珠单抗通过中和血管内皮生长因子（VEGF）用于治疗年龄相关性黄斑变性。本研究旨在研究其对视外血视网膜屏障的潜在副作用。
本研究使用人胎儿 RPE（hfRPE）细胞，因为其在培养中高度分化。通过 RT-PCR、免疫印迹分析和免疫荧光检测 RPE 紧密连接的闭合蛋白组成。ELISA 检测 VEGF 和液相等分子标记物甲基聚乙二醇（mPEG）的分泌和转运。通过跨上皮电阻（TER）衍生的 K+和 Na+电导率（TER）以及 NaCl 和 mPEG 的通量评估紧密连接功能。
在 RPE 紧密连接中检测到闭合蛋白-3、闭合蛋白-10 和闭合蛋白-19。VEGF 以相等的量分泌到顶侧和基底外侧膜，但顶侧膜在摄取和降解 VEGF 方面更活跃。外源性 VEGF 和 mPEG 通过转胞吞作用穿过 RPE 单层，主要是在顶侧到基底的方向。RPE 紧密连接对 K+具有选择性，但对 Na+和 Cl-没有选择性。VEGF、贝伐单抗和雷珠单抗对 TER、mPEG 的通透性以及对 K+、Na+和 Cl-的选择性影响最小。它们对闭合蛋白的表达和分布影响最小。
RPE 具有将 VEGF 浓度维持在视外空间的低浓度的机制，包括内吞作用和降解以及顶侧到基底的液相等分子转胞吞作用。RPE 紧密连接对 K+具有选择性，而对 Na+和 Cl-没有选择性。VEGF、贝伐单抗和雷珠单抗均不影响连接的通透性和选择性。
