Reactive oxygen species induce RNA damage in human atherosclerosis
Reactive oxygen species induce RNA damage in human atherosclerosis
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DOI：
10.1111/j.1365-2362.2004.01343.x
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发表时间：
2004-05-01
影响因子：
5.5
通讯作者：
Kockx, MM
中科院分区：
文献类型：
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作者：
Martinet, W;De Meyer, GRY;Kockx, MM
背景活性氧簇(ROS)诱导的DNA损伤最近在人类和实验性动脉粥样硬化中被发现。本研究旨在探讨动脉粥样硬化斑块中是否存在RNA损伤及其是否与氧化应激有关。材料与方法使用Agilent 2100生物分析仪分析颈动脉内膜切除术标本(n=20)和非动脉粥样硬化性乳动脉(n=20)总RNA的完整性。结果在20个动脉粥样硬化斑块中，有11个斑块的18S/28S rRNA峰明显减少，RNA图谱向短片段方向移动。相反，所有乳腺动脉均显示高质量的RNA，18S和28S rRNA峰清晰可见。氧化损伤标记物7，8-二氢-8-氧代-2‘-鸟苷(8-oxoG)在整个斑块的平滑肌细胞(SMC)、巨噬细胞和内皮细胞均有较强的胞核和胞浆免疫反应，而邻近正常中膜的SMC和乳腺动脉内皮细胞未检测到8-oxoG。当组织切片经RNaseA处理后，斑块中的细胞质8-oxoG染色明显减少，提示RNA的氧化碱基损伤。结论人动脉粥样硬化斑块中存在RNA完整性丧失和8-oxoG氧化修饰。由于RNA损伤可能会影响体外转录本的定量，如果缺乏独立的实验验证(例如，对RNA完整性的评估)，必须谨慎地解释RT-PCR结果。
Background Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress.Materials and methods The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry.Results Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation.Conclusion Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.