内皮型一氧化氮合酶的基因转移改善了高血压大鼠模型中一氧化氮依赖的内皮功能。

Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model.

作者信息

Alexander M Y, Brosnan M J, Hamilton C A, Downie P, Devlin A M, Dowell F, Martin W, Prentice H M, O'Brien T, Dominiczak A F

机构信息

Department of Medicine and Therapeutics, University of Glasgow, UK.

出版信息

Cardiovasc Res. 1999 Aug 15;43(3):798-807. doi: 10.1016/s0008-6363(99)00146-7.

DOI:10.1016/s0008-6363(99)00146-7

PMID:10690352

Abstract

OBJECTIVE

We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function.

METHODS

Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control beta-galactosidase-containing virus, (2 x 10(9) pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo.

RESULTS

Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for beta-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10(-9) to 3 x 10(-6) M) in the absence and presence of NG-nitroarginine methyl ester (100 microM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n = 6 for each; P = 0.0069; 95% CI, 0.864 to 3.277).

CONCLUSIONS

Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.

摘要

目的

我们之前已经表明，与正常血压对照品系Wistar Kyoto（WKY）大鼠相比，人类原发性高血压模型——易卒中型自发性高血压大鼠（SHRSP）的血管内皮水平存在相对一氧化氮缺乏。本研究的目的是调查将内皮型一氧化氮合酶（eNOS）cDNA（AdCMVeNOS）通过腺病毒介导的基因转移至SHRSP的颈动脉是否可改善内皮功能。

方法

使用Griess分析法在培养的内皮细胞中检测AdCMVeNOS编码的重组eNOS蛋白的酶活性。手术分离SHRSP的左颈动脉，并在体外和体内使其暴露于AdCMVeNOS或含对照β-半乳糖苷酶的病毒（2×10⁹ pfu/ml）。术后24小时收获血管，并通过蛋白质免疫印迹法、免疫组织化学以及体外检测内皮功能进行分析。

结果

培养的内皮细胞对两种病毒的转导率几乎均为100%，eNOS表达的剂量反应显示AdCMVeNOS使亚硝酸盐生成增加了五倍，而含β-半乳糖苷酶的病毒则无变化。蛋白质免疫印迹法表明，与对照相比，注入AdCMVeNOS的血管中eNOS表达显著增加。免疫组织化学显示，注入AdCMVeNOS的血管完整内皮细胞中，抗eNOS单克隆抗体染色呈高度阳性。在不存在和存在NG-硝基精氨酸甲酯（100 μM）的情况下，对去氧肾上腺素（10⁻⁹至3×10⁻⁶ M）浓度反应的曲线下面积显示，与对照相比，注入AdCMVeNOS的颈动脉中基础一氧化氮生物利用度增加（每组n = 6；P = 0.0069；95% CI，0.864至3.277）。