Hypoxia/reoxygenation induces CTGF and PAI-1 in cultured human retinal pigment epithelium cells
Hypoxia/reoxygenation induces CTGF and PAI-1 in cultured human retinal pigment epithelium cells
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DOI：
10.1016/j.exer.2008.11.036
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发表时间：
2009-05-01
影响因子：
3.4
通讯作者：
Welge-Lussen, Ulrich
中科院分区：
文献类型：
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作者：
Fuchshofer, Rudolf;Yu, Alice L.;Welge-Lussen, Ulrich
早期年龄相关性黄斑变性（AMD）的特征在于由于细胞外基质（ECM）的积累而导致的布鲁赫膜增厚。这一发现可能与视网膜色素上皮（RPE）缺氧有关。在本研究中，我们研究了缺氧和复氧对结缔组织生长因子（CTGF），纤溶酶原激活物抑制剂-1（派-1），IV型胶原（Col IV）和纤维连接蛋白（Fn）在培养的人视网膜色素上皮细胞的表达的影响。将培养的人RPE细胞在缺氧条件（1%O-2）下保持12-36小时。进行24 h复氧。采用免疫组化、北方和Western印迹分析缺氧介导的CTGF和派-1表达。加入放线菌素D以检测缺氧是否诱导CTGF和派-1 mRNA的转录。此外，用针对缺氧诱导因子-1 α（HIF-1 α）的siRNA转染细胞并保持在缺氧条件下。通过实时荧光定量PCR分析检测抗氧化剂对缺氧/复氧介导的CTGF和派-1表达的影响。通过实时荧光定量PCR和Western印迹分析来研究Col IV和Fn的产生。缺氧和缺氧/复氧均能增加CTGF、派-1、Col IV和Fn的表达。放线菌素D抑制缺氧引起的CTGF和派-1 mRNA的新转录。使用针对HIF-1 α的siRNA，缺氧介导的CTGF和派-1的增加被抑制。抗氧化剂可减弱再氧介导的CTGF和派-1的升高。RPE中缺氧/复氧的过程可能导致RPE中ECM的增加，因此可能有助于ECM在Bruch膜中的积累。(C)2008年由Elsevier Ltd.出版
Early age-related macular degeneration (AMD) is characterized by thickening of Bruch's membrane due to the accumulation of extracellular matrix (ECM). This finding could be related to hypoxia of the retinal pigment epithelium (RPE). In the present study, we investigated the effects of hypoxia and reoxygenation on the expression of connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), collagen type IV (Col IV) and fibronectin (Fn) in cultured human RPE cells. Cultured human RPE cells were kept for 12-36 h under hypoxic conditions (1% O-2). Reoxygenation was conducted for 24 h. Hypoxia-mediated CTGF and PAI-1 expression were analyzed by using immunohistochemistry, Northern and Western blot analysis. Actinomycin D was added to examine whether hypoxia induces the transcription of CTGF and PAI-1 mRNA. Furthermore, cells were transfected with siRNA against hypoxia-inducible factor-1alpha (HIF-1 alpha) and kept under hypoxic conditions. The effects of antioxidants on hypoxia/reoxygenation-mediated CTGF and PAI-1 expression were tested by real-time PCR analysis. Production of Col IV and Fn were investigated by real-time PCR and Western blot analysis. Both hypoxia and hypoxia/reoxygenation increased the expression of CTGF, PAI-1, Col IV and Fn. Actinomycin D prevented the new transcription of CTGF and PAI-1 mRNA by hypoxia. Using siRNA against HIF-1 alpha, the hypoxia-mediated increase of CTGF and PAI-1 was inhibited. Antioxidants attenuated the reoxygenation-mediated increase of CTGF and PAI-1. The process of hypoxia/reoxygenation in the RPE may lead to an increase of ECM in the RPE and thus may contribute to the accumulation of ECM in Bruch's membrane. (C) 2008 Published by Elsevier Ltd.