Tsutsui M, Chen A F, O'Brien T, Crotty T B, Katusic Z S
Department of Anesthesiology, Mayo Clinic, Rochester, Minn 55905, USA.
Arterioscler Thromb Vasc Biol. 1998 Aug;18(8):1231-41. doi: 10.1161/01.atv.18.8.1231.
The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.
本研究旨在确定重组内皮型一氧化氮合酶（eNOS）基因表达对离体犬基底动脉、冠状动脉或股动脉中缓激肽介导的内皮依赖性舒张的影响。将动脉环离体（37℃下30分钟）暴露于编码eNOS基因（AdCMVeNOS）或β-半乳糖苷酶报告基因（AdCMVβ-Gal）的腺病毒载体。转导24小时后，转基因表达主要出现在外膜。重组蛋白在基底动脉中的表达远高于冠状动脉或股动脉。将有内皮和无内皮的对照、AdCMVβ-Gal和AdCMVeNOS动脉环悬挂起来进行等长张力记录。通过放射免疫测定法测量cGMP水平。在有内皮的AdCMVeNOS基底动脉中，对低浓度缓激肽（3×10⁻¹¹至10⁻⁹mol/L）的舒张作用显著增强。相反，在有内皮的冠状动脉和股动脉中，AdCMVeNOS转导不影响对缓激肽的舒张反应。去除内皮后，对照和AdCMVβ-Gal基底动脉中缓激肽诱导的舒张作用消失。然而，在用AdCMVeNOS转导的基底动脉中，即使去除内皮，用缓激肽（3×10⁻¹¹至10⁻⁹mol/L）刺激仍会引起舒张以及cGMP生成增加。缓激肽诱导的舒张作用被一氧化氮合酶抑制剂NG-硝基-L-精氨酸甲酯完全阻断。电子显微镜分析显示，重组eNOS蛋白在外膜的成纤维细胞中表达。这些结果表明，基因修饰的外膜成纤维细胞可能恢复无内皮脑动脉中一氧化氮的生成。我们的研究结果支持血管生物学中的一个新概念，即外膜中的成纤维细胞在重组eNOS基因成功转移和表达后可能参与血管张力的调节。
