重组内皮型一氧化氮合酶基因在猪冠状动脉中的表达及功能

Cable D G, O'Brien T, Kullo I J, Schwartz R S, Schaff H V, Pompili V J

Cardiac Surgical Research Center, Mayo Clinic, Rochester, MN, USA.

Cardiovasc Res. 1997 Sep;35(3):553-9. doi: 10.1016/s0008-6363(97)00161-2.

OBJECTIVES

Direct gene transfer of exogenous nitric oxide synthase, with the subsequent increase in nitric oxide production, could represent a potential therapeutic strategy in the treatment of vascular proliferative disorders. The goal of the present study was to determine if porcine coronary arteries could be transduced with an adenoviral vector encoding endothelial nitric oxide synthase (Ad.CMVeNOS) resulting in functional expression.

METHODS AND RESULTS

Segments of porcine right coronary artery were exposed for 1 h at 37 degrees C to either replication-deficient adenovirus encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS, 5 x 10(9) pfu/ml) or control adenovirus encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ, 5 x 10(9) pfu/ml). Immunohistochemistry with a monoclonal antibody specific for nitric oxide synthase (NOS) demonstrated recombinant gene expression in both the endothelial and adventitial layers of Ad.CMVeNOS transduced coronaries with only endogenous NOS confirmed in the endothelium of Ad.CMVLacZ arteries. Coronary arteries transduced with Ad.CMVeNOS yielded 517 +/- 110 (mean +/- S.E.M.) nM/ng nitrite while vessels transduced with Ad.CMVLacZ yielded 126 +/- 84 nM/ng (P < 0.05, n = 6). Isometric tension recording, following prostaglandin F2 alpha constriction, documented a reduced tension in Ad.CMVeNOS transduced coronaries, compared to matched Ad.CMVLacZ coronaries (6.10 +/- 1.08 g vs. 8.45 +/- 1.19 g, respectively, P = 0.05, n = 8). This tension differential was eliminated with prior incubation in NG-monomethyl-L-arginine (L-NMMA, 10(-4) M). The EC50 for calcium ionophore relaxation of Ad.CMVeNOS coronary arteries was reduced compared to Ad.CMVLacZ (-7.90 +/- 0.03 logM vs. -7.26 +/- 0.11 logM, respectively, P < 0.05, n = 8).

CONCLUSIONS

These studies demonstrate successful transfer of endothelial nitric oxide synthase into porcine coronary arteries as verified by histochemical localization of recombinant protein with an increase of nitric oxide release as demonstrated by enhanced nitrite production and an alteration in vasomotor function.

目的

直接导入外源性一氧化氮合酶基因，随后增加一氧化氮的生成，可能是治疗血管增殖性疾病的一种潜在治疗策略。本研究的目的是确定编码内皮型一氧化氮合酶的腺病毒载体（Ad.CMVeNOS）能否转导猪冠状动脉并实现功能性表达。

方法与结果

将猪右冠状动脉段在37℃下暴露于编码牛内皮型一氧化氮合酶的复制缺陷型腺病毒（Ad.CMVeNOS，5×10⁹ pfu/ml）或编码大肠杆菌β-半乳糖苷酶的对照腺病毒（Ad.CMVLacZ，5×10⁹ pfu/ml）1小时。用针对一氧化氮合酶（NOS）的单克隆抗体进行免疫组织化学检测，结果显示Ad.CMVeNOS转导的冠状动脉的内皮和外膜层均有重组基因表达，而Ad.CMVLacZ动脉的内皮中仅证实有内源性NOS。Ad.CMVeNOS转导的冠状动脉产生517±110（平均值±标准误）nM/ng亚硝酸盐，而Ad.CMVLacZ转导的血管产生126±84 nM/ng（P<0.05，n = 6）。在前列腺素F2α收缩后进行等长张力记录，结果表明与匹配的Ad.CMVLacZ冠状动脉相比，Ad.CMVeNOS转导的冠状动脉张力降低（分别为6.10±1.08 g和8.45±1.19 g，P = 0.05，n = 8）。在NG-单甲基-L-精氨酸（L-NMMA，10⁻⁴ M）中预先孵育可消除这种张力差异。与Ad.CMVLacZ相比，Ad.CMVeNOS冠状动脉对钙离子载体舒张的EC50降低（分别为-7.90±0.03 logM和-7.26±0.11 logM，P<0.05，n = 8）。