Ooboshi H, Toyoda K, Faraci F M, Lang M G, Heistad D D
Departments of Internal Medicine and Pharmacology (F.M.F., D.D.H.), Cardiovascular Center and Center on Aging, University of Iowa College of Medicine, Iowa City, USA.
Arterioscler Thromb Vasc Biol. 1998 Nov;18(11):1752-8. doi: 10.1161/01.atv.18.11.1752.
Gene transfer with replication-deficient adenovirus is a useful tool to study vascular biology. We have reported that overexpression of endothelial nitric oxide (NO) in carotid arteries from normal rabbits augments vasorelaxation mediated by NO. In this study, we tested the hypothesis that adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) improves impaired relaxation of atherosclerotic vessels. We used 2 replication-deficient adenoviruses: AdeNOS, which carries cDNA for eNOS, and Adbetagal, which expresses beta-galactosidase. Common carotid arteries from 10 New Zealand White (NZW; plasma cholesterol, 79+/-13 mg/dL) and 10 Watanabe heritable hyperlipidemic (WHHL; plasma cholesterol, 452+/-39 mg/dL) rabbits were incubated in organ culture with AdeNOS, Adbetagal, or vehicle alone. Carotid arteries from WHHL rabbits had mild to moderate atherosclerotic lesions. Histochemical staining for beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in the endothelium and adventitia in both NZW and WHHL rabbits. Expression of eNOS determined with Western blot analysis after incubation with AdeNOS tended to be higher in vessels from WHHL rabbits than NZW rabbits. Effects of transgene expression on vascular function were examined by recording isometric tension 1 day after transduction. After precontraction with phenylephrine, acetylcholine produced significantly less relaxation in vessels from WHHL rabbits than in vessels from NZW rabbits. Relaxation in response to acetylcholine was greater in carotid arteries from both NZW and WHHL rabbits that were transfected with AdeNOS than in vessels treated with vehicle or Adbetagal. Vasorelaxation in response to acetylcholine was inhibited by Nomega-nitro-L-arginine. Responses to sodium nitroprusside were similar after treatment with vehicle alone, Adbetagal, or AdeNOS in both groups of rabbits. Thus, overexpression of eNOS with an adenoviral vector improves impaired NO-mediated relaxation in atherosclerotic arteries.
利用复制缺陷型腺病毒进行基因转移是研究血管生物学的一种有用工具。我们曾报道，正常兔颈动脉中内皮型一氧化氮（NO）的过表达增强了由NO介导的血管舒张。在本研究中，我们检验了以下假设：腺病毒介导的内皮型一氧化氮合酶（eNOS）基因转移可改善动脉粥样硬化血管受损的舒张功能。我们使用了两种复制缺陷型腺病毒：携带eNOS cDNA的AdeNOS和表达β-半乳糖苷酶的Adbetagal。将10只新西兰白兔（NZW；血浆胆固醇，79±13 mg/dL）和10只渡边遗传性高脂血症兔（WHHL；血浆胆固醇，452±39 mg/dL）的颈总动脉在器官培养中分别与AdeNOS、Adbetagal或单独的载体一起孵育。WHHL兔的颈动脉有轻度至中度动脉粥样硬化病变。β-半乳糖苷酶的组织化学染色和eNOS的免疫组织化学表明，在NZW和WHHL兔的内皮和外膜中均有转基因表达。用AdeNOS孵育后，通过蛋白质印迹分析测定的eNOS表达在WHHL兔的血管中往往高于NZW兔。在转导1天后，通过记录等长张力来检查转基因表达对血管功能的影响。用去氧肾上腺素预收缩后，乙酰胆碱在WHHL兔血管中产生的舒张作用明显小于在NZW兔血管中产生的舒张作用。在NZW和WHHL兔中，转染AdeNOS的颈动脉对乙酰胆碱的舒张反应均大于用载体或Adbetagal处理的血管。Nω-硝基-L-精氨酸抑制了对乙酰胆碱的血管舒张反应。在两组兔中，单独用载体、Adbetagal或AdeNOS处理后，对硝普钠的反应相似。因此，用腺病毒载体使eNOS过表达可改善动脉粥样硬化动脉中受损的NO介导的舒张功能。
