Hypoxia inducible factor-1 activation by prolyl 4-hydroxylase-2 gene silencing attenuates myocardial ischemia reperfusion injury
Hypoxia inducible factor-1 activation by prolyl 4-hydroxylase-2 gene silencing attenuates myocardial ischemia reperfusion injury
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DOI：
10.1161/01.res.0000197816.63513.27
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发表时间：
2006-01-06
影响因子：
20.1
通讯作者：
Fowler, AA
中科院分区：
文献类型：
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作者：
Natarajan, R;Salloum, FN;Fowler, AA
缺氧诱导因子-1（HIF-1）调节缺氧/缺血环境中关键基因如诱导型一氧化氮合酶（iNOS）转录的变化。在常氧条件下，HIF-1的活化受HIF-1 α-脯氨酰4-羟化酶控制，该酶靶向HIF-1 α进行泛素化和蛋白酶体降解。我们假设常氧HIF-1保存可以通过预处理效应减轻心脏缺血/再灌注损伤。通过使用小干扰RNA（siRNA）沉默小鼠HIF-1 α-脯氨酰-4羟化酶-2（PHD 2）来实现HIF-1的保存。在常氧小鼠微血管内皮细胞（EC）中，PHD 2 siRNA以时间和浓度依赖性方式使PHD 2 mRNA表达降低89 +/- 1.5%（P < 0.001）。常氧EC中PHD 2沉默稳定了HIF-1 α蛋白水平，同时显著增加了HIF-1转录活性和iNOS mRNA表达。输注PHD 2 siRNA（1.5 μ g/g体重）的野生型小鼠在24小时内显示心脏PHD 2 mRNA减少61 +/- 2.4%（P < 0.05）。此外，HIF-1 α蛋白水平和HIF-1依赖性iNOS mRNA水平增加。与盐水处理的对照相比，来自经受30分钟缺血随后60分钟再灌注的野生型小鼠（n=6）的PHD 2 siRNA转染的心脏表现出减小的梗死面积（分别为9.7 +/- 1.9%对31.6 +/-1.8%，P < 0.0001，n=6）和用非靶向siRNA对照转染的对照小鼠（28.4 +/-3.0%，P < 0.0001，n=6）。来自通过相同注射方案接受PHD 2 siRNA的iNOS敲除小鼠（n=6）的心脏显示出与盐水对照（28.7 +/-1.3%）不可区分的梗死面积。这些结果表明，在体外和体内，使用siRNA策略的PHD 2沉默产生转录活性HIF-1。体内PHD 2 siRNA给药后心脏中HIF-1的常氧激活通过iNOS依赖性途径减轻再灌注损伤。
Hypoxia inducible factor-1 (HIF-1) regulates changes in transcription of key genes such as inducible NO synthase (iNOS) in hypoxic/ischemic environments. In normoxia, HIF-1 activation is controlled by HIF-1 alpha-prolyl 4-hydroxylases, which target HIF-1 alpha for ubiquitination and proteasomal degradation. We hypothesized that normoxic HIF-1 preservation could attenuate cardiac ischemia/reperfusion injury via a preconditioning effect. HIF-1 preservation was achieved by using small interfering RNA (siRNA) to silence murine HIF-1 alpha-prolyl-4hydroxylase-2 (PHD2). PHD2 siRNA reduced PHD2 mRNA expression 89 +/- 1.5% (P < 0.001) in a time- and concentration-dependent manner in normoxic murine microvascular endothelial cells (EC). PHD2 silencing in normoxic EC stabilized HIF-1 alpha protein levels while significantly increasing HIF-1 transcriptional activity and iNOS mRNA expression. Wild-type mice infused with PHD2 siRNA (1.5 mu g/g body weight) showed a 61 +/- 2.4% (P < 0.05) reduction in cardiac PHD2 mRNA within 24 hours. In addition HIF-1 alpha protein levels and HIF-1-dependent iNOS mRNA levels were increased. PHD2 siRNA-transfected hearts from wild-type mice (n=6) subjected to 30 minutes ischemia followed by 60 minutes reperfusion exhibited reduced infarct size when compared with saline-treated controls (9.7 +/- 1.9% versus 31.6 +/- 1.8%, respectively, P < 0.0001, n=6) and to control mice transfected with a nontargeting siRNA control (28.4 +/- 3.0%, P < 0.0001, n=6). Hearts from iNOS knockout mice receiving PHD2 siRNA by identical injection protocol (n=6) exhibited infarct size indistinguishable from saline controls (28.7 +/- 1.3%). These results show that in vitro and in vivo, PHD2 silencing using a siRNA strategy produces transcriptionally active HIF-1. Normoxic activation of HIF-1 in hearts following in vivo PHD2 siRNA administration attenuates reperfusion injury via an iNOS-dependent pathway.