Balance-shift in the production of NO and superoxide via endothelial nitric oxide synthase in atherogenesis

内皮型一氧化氮合酶(ENOS)被认为是抗动脉粥样硬化的分子。事实上，最近对eNOS基因缺陷小鼠的研究表明，缺乏eNOS基因的小鼠动脉粥样硬化病变形成加速。另一方面，研究发现，在NO合成的重要辅助因子四氢生物蝶呤(BH4)缺乏或缺乏eNOS酶活性的情况下，eNOS功能障碍，产生超氧化物而不是NO。这种现象被称为eNOS解偶联。在目前的研究中，我们研究了eNOS过度表达如何影响高胆固醇血症下的动脉粥样硬化形成。我们将载脂蛋白E基因敲除小鼠(apo E-KO)与内皮细胞过度表达eNOS的eNOS转基因小鼠(eNOS-TG)进行杂交。我们发现在高胆固醇血症下，apo E-KO/eNOS-TG组较apo E-KO组动脉超氧化物歧化产物增多，而NO生成量相对较低。因此，推测载脂蛋白E-KO/eNOS-TG中存在eNOS解偶联现象。我们还发现eNOS的过表达加速了载脂蛋白E-KO动脉粥样硬化病变的形成。载脂蛋白E-KO/eNOS-TG组血管BH4水平低于载脂蛋白E-KO组。外源性BH4慢性治疗apo E-KO/eNOS-TG可减少动脉超氧化物歧化产物的生成，增加动脉血中NO的生成，缩小动脉粥样硬化病变面积。进一步过表达BH4合成的限速酶GTPCH1与GTPCH1转基因小鼠杂交，也可减少载脂蛋白E-KO/eNOS-TG的动脉粥样硬化病变面积。因此，在血管组织中BH4水平和eNOS蛋白水平之间似乎存在着微小的平衡。当BH4水平在高胆固醇血症下降低时，eNOS解偶联，产生超氧化物而不是NO，这有助于加速动脉粥样硬化的形成。

NO from the endothelial NO synthase (eNOS) is believed to act as anti-atherogenic molecule. Indeed recent studies in eNOS gene-deficient mice showed the accelerated atherosclerotic lesion formation in mice that lacks eNOS gene. On the other hand, it is revealed that eNOS becomes dysfunctional and produces superoxide rather than NO under conditions in which tetrahydrobiopterin (BH4), an essential cofactor for NO synthesis, is lacking or deficient for eNOS enzymatic activity. This phenomenon is called eNOS uncoupling. In the present study we examined how eNOS overexpression affects atherogenesis under hypercholesterolemia. We crossed apo E-knockout mice (apo E-KO) with eNOS transgenic mice that overexpress eNOS in the endothelium (eNOS-Tg). We found that under hypercholesterolemia superoxide production was increased and NO production was relatively low in the aortas from apo E-KO/eNOS-Tg compared with those from apo E-KO. Therefore the presence of eNOS uncoupling was suggested in apo E-KO/eNOS-Tg. We also revealed that eNOS overexpression accelerated atherosclerotic lesion formation in apo E-KO. Vascular levels of BH4 were decreased in apo E-KO/eNOS-Tg compared with apo E-KO. Chronic treatment of apo E-KO/eNOS-Tg with exogenous BH4 decreased superoxide production and increased NO production from aortas, and reduced the atherosclerotic lesion area. Further overexpression of GTPCH 1, the rate limiting enzyme of BH4 synthesis, by crossing with GTPCH 1 transgenic mice also decreased atherosclerotic lesion area in apo E-KO/eNOS-Tg. Therefore, it seems that there is a minute balance between BH4 levels and eNOS protein levels in vascular tissue. When BH4 levels decrease under hypercholesterlemia, eNOS becomes uncoupling and produces superoxide rather than NO, which serves to accelerate atherogenesis.