Oxidative stress affects the junctional integrity of retinal pigment epithelial cells
Oxidative stress affects the junctional integrity of retinal pigment epithelial cells
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DOI：
10.1167/iovs.03-0351
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发表时间：
2004-02-01
影响因子：
4.4
通讯作者：
Cheetham, ME
中科院分区：
文献类型：
--
作者：
Bailey, TA;Kanuga, N;Cheetham, ME
目的.氧化应激与年龄相关性黄斑变性的发病机制有关。因此，检测了细胞系ARPE-19对氧化应激的反应及其对应激蛋白诱导和连接完整性的影响。响应于不同浓度的过氧化氢，评估培养1周或5周后的ARPE-19细胞活力。通过免疫荧光和Western印迹分析检测血红素氧合酶（HO）-1，Hsp27和Hsp70来评估对亚致死剂量的反应。免疫荧光法用于研究连接蛋白封闭小带（ZO）-1，occludin，N-cadherin和β-catenin的定位。亚细胞分级分离用于评估β-连环蛋白的任何再分布。结果培养5周的ARPE-19细胞对过氧化氢诱导的慢性氧化应激的敏感性低于培养1周的细胞。更分化的ARPE-19细胞具有更高的Hsp27和Hsp70稳态水平。在文化中，对压力的反应也随时间而不同。连接蛋白的定位，这成为强烈的周边培养5周后，成为破坏后氧化应激，和胞浆β-连环蛋白增加。慢性氧化应激也增加了跨单层的细胞旁流量。在ARPE-19细胞中，随着分化，对慢性氧化应激的抗性增加与Hsp27和Hsp70的稳态水平较高相关。氧化应激破坏RPE细胞连接和屏障完整性，这可能通过破坏血-视网膜屏障而促成与RPE相关的疾病的发病机制。
PURPOSE. Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration. The cell line ARPE-19 was therefore examined for response to oxidative stress and its effect on stress protein induction and junctional integrity.METHODS. ARPE-19 cell viability after 1 week or 5 weeks in culture was assessed in response to different concentrations of hydrogen peroxide. The response to sublethal doses was assessed by examination of heme oxygenase (HO)-1, Hsp27 and Hsp70 by immunofluorescence and Western blot analysis. Immumofluorescence was used to investigate the localization of the junctional proteins zonula occludens (ZO)-1, occludin, and N-cadherin, and beta-catenin. Subcellular fractionation was used to assess any redistribution of beta-catenin. Monolayer integrity was examined by measurement of flux of rhodamine-conjugated dextrans from the apical to basal aspect of cells.RESULTS ARPE-19 cells cultured for 5 weeks were less sensitive to chronic oxidative stress induced by hydrogen peroxide than those cultured for 1 week. The more differentiated ARPE-19 cells had higher steady state levels of Hsp27 and Hsp70. The response to stress also differed with time in culture. The localization of junctional proteins, which became strongly peripheral after 5 weeks in culture, became disrupted after oxidative stress, and cytosolic beta-catenin increased. Chronic oxidative stress also increased paracellular flux across the monolayer.CONCLUSIONS. Increased resistance to chronic oxidative stress with differentiation in ARPE-19 cells correlated with higher steady state levels of Hsp27 and Hsp70. Oxidative stress disrupted RPE cell junction and barrier integrity, which may contribute to the pathogenesis of diseases related to RPE through disruption of the blood-retinal barrier.