Molecular analyses of hypoxia response of pulmonary arterial endothelium by using genetic engineering

[目的]缺氧诱导因子I（Hypoxia-inducible factor I，HW-1）最初被鉴定为缺氧诱导的核因子，与促红细胞生成素（Erythropoietin，Epo）的缺氧反应元件（hypoxia-response element，HRE）结合。HIF-1通过激活基因的转录在O2稳态中起重要作用，所述基因的产物介导对缺氧的必需细胞和全身反应，包括红细胞生成、糖酵解、血管发生和血管舒张。本研究的目的是阐明肺动脉（PA）内皮细胞的缺氧反应，通过添加基因修饰，分泌报告基因（Re）产物后，腺病毒介导的RecDNA和缺氧反应顺式元件。[方法]构建两种腺病毒载体，观察缺氧对肺动脉内皮细胞的影响。一种腺病毒载体含有从Epo基因组分离的具有缺氧反应顺式元件（HRE）的Re表达盒（AdHRE），另一种腺病毒载体含有不具有缺氧反应顺式元件的Re表达盒（AdHRE）。 ...更多信息 HRE（Ad）作为对照载体。利用这些载体，以缺氧和常氧时Re产物的生成比率来评价PA内皮、主动脉内皮的缺氧反应。[结果]与常氧条件下Ad处理的内皮细胞相比，缺氧条件下Ad处理的内皮细胞Re生成率无明显增加。与此相反，AdHRE感染的PA内皮细胞在缺氧条件下的Re生成率比常氧条件下的增加了4倍（p<0.001）。此外，有趣的是，暴露于缺氧的远端PA中的AdHRE感染的内皮细胞具有显著大的Re产生比率增加，与暴露于缺氧的近端PA中的AdHRE感染的内皮细胞相比增加了2倍（p<0.001）。为研究不同类型内皮细胞的缺氧反应，将主动脉内皮细胞缺氧后，AdHRE处理的主动脉内皮细胞在缺氧12小时后，其Re生成率出现一个平台期，是常氧下AdHRE处理的内皮细胞的4倍或2倍。[结论]利用腺病毒介导的缺氧反应顺式元件，建立了缺氧反应分析系统。本实验结果表明，肺动脉栓塞的缺氧反应明显大于体循环栓塞，尤其是与血管重构相关的肺动脉远端内皮细胞的缺氧反应更明显。HRE基因转移系统可用于评价循环系统或肺系统疾病的缺氧反应障碍。少

[Purpose] Hypoxia-inducible factor I (HW-1) is originally identified as a nuclear factor that is induced by hypoxia and bound to a site in the erythropoietin (Epo) hypoxia-response element (HRE). HIF-1 plays an important role in 02 homeostasis by activating transcription of genes whose' products mediate essential cellular and systemic response to hypoxia, including erythropoiesis, glycolysis, vasculogenesis, and vasodilatation. The aim of this study is to clarify hypoxia response of pulmonary arterial (PA) endothelium by addition of genetic modification to secrete reporter gene (Re) product following adenovirus-mediated transfer of both Re cDNA and hypoxia response cis-element. [Method] To evaluate hypoxia response of PA endothelium, two type of adenovirus vector were constructed. One adenovirus vector contains Re expression cassette with hypoxia response cis element (HRE) isolated from Epo genome (AdHRE), and the other one is adenovirus vector containing Re expression cassette without … More HRE (Ad) as control vector. Using these vectors, hypoxia response of PA endothelium, aortic endothelium was evaluated by using the ratio of production of Re product in exposure to hypoxia and normoxia. [Results) Each Ad treated-endothelium exposed to hypoxia had no increase in the ratio of Re production, compared with Ad treated endothelium in normoxia. In marked contrast, AdHRE-infected PA endothelium exposed to hypoxia had an increase in the ratio of Re production, 4 fold compared with AdHRE infected PA endothelium in normoxia (p<O.OOl). Furthermore, interestingly, AdHRE infected endothelium in distal PA exposed to hypoxia had a significantly large increase in the ratio of Re production, 2 fold compared with AdHRE infected endotbeliuxn in proximal PA exposed to hypoxia (p<O.OOl). To evaluate hypoxia response in different type of endothelium, aortic endothelium was exposed to hypoxia following treatment with AdHERE.AdHRE-treated-aortic endothelium has a plateau in the ratio of Re production 12 hrs following the exposure to hypoxia, 4 or 2 fold compared with AdHRE-treated each endothelium in normoxia. [Conclusion] Analysis system of hypoxia response was established by using adenovirus-mediated transfer of hypoxia response cis element. Our current study using this HRE transfer system demonstrated that hypoxia response of PAE showed larger than that of AE of systemic circulation, especially endothelium in distal PA relevant to vascular remodeling had a markedly larger hypoxia response. This HRE- gene transfer system was feasible to evaluate disorder of hypoxia response in circulatory or pulmonary diseases. Less