Kullo I J, Schwartz R S, Pompili V J, Tsutsui M, Milstien S, Fitzpatrick L A, Katusic Z S, O'Brien T
Division of Cardiovascular Disease, Mayo Clinic, Rochester, MN 55905, USA.
Arterioscler Thromb Vasc Biol. 1997 Nov;17(11):2405-12. doi: 10.1161/01.atv.17.11.2405.
Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosidase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.
平滑肌细胞（SMCs）在血管疾病的发病机制中起关键作用。本研究的目的是确定将重组内皮型一氧化氮合酶（eNOS）基因转移到猪冠状动脉平滑肌细胞（CSMCs）是否会导致功能性酶的表达，并评估eNOS表达对细胞增殖的影响。用编码eNOS（AdeNOS）和β-半乳糖苷酶（AdβGal）cDNA的腺病毒载体在体外转导CSMCs。与AdβGal转导或假转导的细胞相比，用AdeNOS转导的CSMCs经NADPH-黄递酶染色呈阳性，获得了钙依赖性NOS活性（通过[3H]L-精氨酸转化为[3H]L-瓜氨酸来测定），随着载体浓度的增加，环3',5'-cGMP水平升高，并且亚硝酸盐产量增加。通过增加eNOS辅因子四氢生物蝶呤的细胞内水平，增强了AdeNOS转导细胞的cGMP产生。通过胸苷摄取测定，用AdeNOS转导的CSMCs显示血清刺激的DNA合成减少。通过对静止CSMCs进行血清刺激后3天和6天的细胞计数评估，AdeNOS转导的CSMCs中的细胞增殖减少。本研究表明，腺病毒介导的eNOS基因转移到CSMCs导致功能性酶的表达，其活性可通过增加细胞内四氢生物蝶呤水平来增强。重组eNOS在CSMCs中的表达导致血清刺激的DNA合成和细胞增殖受到抑制。这些发现意味着eNOS基因转移到SMCs可能是增加动脉壁局部NO产生的独特方式。
