Effects Of Nitric Oxide Inhalation On Response To Vascul

一氧化氮调节血管损伤的新生内膜反应，如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所示。相反，缺乏编码内皮型一氧化氮合酶基因的小鼠对血管损伤的新生内膜反应增强。最近，已经描述了一氧化氮在血液中的转运，提出与血红蛋白分子（S-亚硝基血红蛋白）的β链的半胱氨酸-93结合的一氧化氮在从肺到外周的这种转运机制中可能特别重要。我们提出了一种假说，即血管损伤后吸入一氧化氮可能会增加一氧化氮在血液中的转运，并通过抑制平滑肌细胞增殖来减少新生内膜的形成。为此，在本实验中研究了20只小鼠：10只C57 BL/6 J野生型小鼠和10只p27敲除小鼠，这些小鼠缺乏编码细胞周期蛋白依赖性激酶抑制剂p27的基因，并且对血管损伤具有增强的新生内膜反应。通过左腿前外侧表面上的纵向切口暴露每只小鼠的股动脉和隐动脉。将一根0.010英寸的柔性导丝引入隐动脉，推进至左股动脉约1.5 cm，然后缩回并再次推进共5次，以产生内膜损伤。右股动脉保持完整并作为对照。将每种品系的5只小鼠置于室内空气中，并将每种品系的5只小鼠置于密闭室中，在密闭室中它们吸入30 ppm一氧化氮和20.9%氧气（用氮气平衡）。在任一环境中14天后，将小鼠安乐死，并收获左股动脉和右股动脉用于形态测定分析。通过直接心脏采血棒获得血液样本，以测量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珠蛋白链的半胱氨酸-93的反应产物）和亚硝基（Fe II）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基因敲除小鼠吸入一氧化氮14天后，高铁血红蛋白和硝酸盐含量比空气动物增加约10倍，这与肺循环中一氧化氮与氧合血红蛋白的反应一致。在吸入一氧化氮后，野生型和p27基因敲除小鼠的亚硝酰（Fe Ⅱ）血红蛋白比室内空气值增加了100倍以上。然而，S-亚硝基血红蛋白水平不变，吸入一氧化氮与动物在室内空气中相比。损伤和未损伤动脉的形态测量显示p27缺失和野生型小鼠之间没有显著差异，并且一氧化氮对损伤的新生内膜反应没有影响，尽管每组动物的数量很少（n=5）。我们的结论是，吸入一氧化氮2周增加了亚硝酰（FeII）血红蛋白的水平，这可能是一个重要的机制，一氧化氮运输从肺循环到全身血管系统。然而，吸入一氧化氮并没有改变S-亚硝基血红蛋白的水平，其水平较低，因此可能不是一氧化氮的重要转运区域血管床。

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.