Effects Of Nitric Oxide Inhalation On Response To Vascul

使用一氧化氮供体药物和内皮一氧化氮合酶基因治疗的动物模型显示，一氧化氮调节血管损伤的新内膜反应。相反，缺乏编码内皮一氧化氮合酶基因的小鼠对血管损伤的新内膜反应增强。最近，血液中一氧化氮的运输已被描述，并提出一氧化氮与血红蛋白分子（s -亚硝基血红蛋白）β链的半胱氨酸-93的结合可能在这种从肺部到外周的运输机制中特别重要。我们提出假设，血管损伤后吸入一氧化氮可能通过抑制平滑肌细胞增殖来增加血液中一氧化氮的运输并减少新内膜的形成。为此，本实验研究了20只小鼠：10只C57BL/6J野生型小鼠和10只p27敲除小鼠，这些小鼠缺乏编码周期蛋白依赖性激酶抑制剂p27的基因，对血管损伤的新内膜反应增强。通过左腿前外侧表面的纵向切口暴露每只小鼠的股动脉和隐动脉。将一根0.010英寸的软丝插入隐动脉，向前推进至左股动脉约1.5 cm，共收进5次，造成内膜损伤。右股动脉保持完整，作为对照。每个品系5只小鼠置于室内空气中，每个品系5只小鼠置于密闭室中，吸入30ppm的一氧化氮和20.9%的氧与氮平衡。在任意一种环境下生活14天后，对小鼠实施安乐死，取左、右股动脉进行形态计量学分析。采用直接心棒法采集血样，测定高铁血红蛋白和硝酸盐（一氧化氮与氧合血红蛋白的反应产物）、s -亚硝基血红蛋白（一氧化氮与β -球蛋白链上的半胱氨酸- 93的反应产物）和亚硝基血红蛋白（一氧化氮与脱氧血红蛋白的反应产物）。我们发现，野生型和p27敲除小鼠在吸入一氧化氮14天后，高铁血红蛋白和硝酸盐的含量比空气动物增加了大约10倍，这与一氧化氮与肺循环中的氧合血红蛋白的反应一致。在这段时间的一氧化氮吸入后，野生型和p27敲除小鼠的亚硝基（FeII）血红蛋白比室内空气值增加了100倍以上。然而，与室内空气中的动物相比，吸入一氧化氮后，s -亚硝基血红蛋白水平没有变化。损伤和未损伤动脉的形态测量结果显示，p27缺失小鼠与野生型小鼠之间没有显著差异，一氧化氮也没有影响新生内膜对损伤的反应，尽管每组动物的数量都很小（n=5）。我们得出结论，吸入一氧化氮2周会增加亚硝基（FeII）血红蛋白水平，这可能是一氧化氮从肺循环转运到全身血管系统的重要机制。然而，吸入一氧化氮并没有改变s -亚硝基血红蛋白的水平，其水平很低，因此可能不是一氧化氮向区域血管床的重要转运体。

Nitric oxide modulates the neointimal response to vascular injury, as shown in animal models using nitric oxide donor drugs and gene therapy with endothelial nitric oxide synthase. Conversely, mice that lack the gene encoding endothelial nitric oxide synthase have enhanced neointimal response to vascular injury. Recently, nitric oxide transport in blood has been described, with the proposal that nitric oxide binding to cysteine-93 of the beta chains of the hemoglobin molecule (S-nitrosohemoglobin) may be of particular importance in this transport mechanism from the lungs to the periphery. We proposed the hypothesis that nitric oxide inhalation following vascular injury may increase nitric oxide transport in blood and reduce neointima formation by inhibiting smooth muscle cell proliferation. To this end, 20 mice were studied in this experiment: 10 C57BL/6J wild-type mice and 10 p27 knock-out mice which lack the gene encoding the cyclin-dependent kinase inhibitor p27 and have an enhanced neointimal response to vascular injury. The femoral and saphenous arteries of each mouse were exposed via a longitudinal incision on the anterolateral surface of the left leg. A 0.010-inch flexible wire was introduced into the saphenous artery, advanced to the left femoral artery about 1.5 cm, and then retracted and re-advanced a total of 5 times to produce intimal injury. The right femoral artery was left intact and served as control. Five mice of each strain were placed in room air and 5 mice of each strain were placed in an airtight chamber where they inhaled 30 ppm nitric oxide and 20.9 % oxygen balanced with nitrogen. After 14 days in either environment, mice were euthanized and the left and right femoral arteries were harvested for morphometric analysis. Blood samples were obtained by a direct heart stick to measure methemoglobin and nitrate (reactant products of nitric oxide with oxyhemoglobin), S-nitrosohemoglobin (reaction product of nitric oxide with cysteine- 93 of the beta globin chain) and nitrosyl(FeII)hemoglobin (reactant product of nitric oxide with deoxyhemoglobin). We found that wild-type and p27 knock-out mice who received nitric oxide inhalation for 14 days had an approximately 10-fold increase in methemoglobin and nitrate over rom air animals, consistent with the reaction of nitric oxide with oxyhemoglobin in the pulmonary circulation. Nitrosyl(FeII)hemoglobin increased over 100-fold over room air values in wild-type and p27 knock-out mice after this priod of nitric oxide inhalation. However, levels of S-nitrosohemoglobin were unchanged with nitric oxide inhalation compared with animals in room air. Morphometric measurements of injured and uninjured arteries showed no significant difference between p27 deletion and wild type mice, and no effect of nitric oxide on the neointimal response to injury, although the numbers of animals in each group was small (n=5). We conclude that nitric oxide inhalation for 2 weeks increases levels of nitrosyl(FeII)hemoglobin, which may represent an important mechanism of nitric oxide transport from the pulmonary circulation to the systemic vasculature. However, nitric oxide inhalation did not change levels of S-nitrosohemoglobin, levels of which were low and thus may not be an important transporter of nitric oxide to regional vascular beds.