Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase
Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase
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DOI：
10.1152/ajplung.00408.2005
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发表时间：
2006-06-01
影响因子：
4.9
通讯作者：
Black, SM
中科院分区：
文献类型：
--
作者：
Grobe, AC;Wells, SM;Black, SM
虽然已知氧化应激导致内皮功能障碍相关的全身性血管疾病，但其在肺血管疾病中的作用尚不清楚。我们以前的研究，使用孤立的肺动脉取自手术造成的心脏缺损和肺血流量增加（分流）的羔羊，已经提出了活性氧（ROS）在肺动脉高压的内皮功能障碍中的作用，但缺乏体内数据。因此，本研究的最初目的是确定分流羔羊是否具有升高的ROS生成水平，以及这是否与抗氧化能力的改变相关。我们的研究结果表明，超氧化物，但不是过氧化氢，水平显着升高分流羔羊。此外，我们发现超氧化物生成的增加与抗氧化酶表达或活性的改变无关。这些数据表明，有一个超氧化物生成的增加，而不是在肺清除能力的下降。因此，我们接下来检查了NADPH氧化酶复合物的各种亚基的表达作为超氧化物产生的潜在来源。结果表明，Rac 1和p47（phox）在分流羔羊中的表达增加。我们还发现，NADPH氧化酶抑制剂二苯基碘（DPI）显着减少二氢乙锭（DHE）氧化制备的肺切片分流，但不控制羔羊。由于DPI也可以抑制内皮型一氧化氮合酶（eNOS）超氧化物的产生，我们使用更特异性的NADPH氧化酶抑制剂（夹竹桃苷）和NOS抑制剂（3-乙基异硫脲）重复了该实验。我们的研究结果表明，这两种抑制剂显着减少DHE氧化肺切片制备的分流，但不控制羔羊。为了进一步研究eNOS在分流羔羊中变得解偶联的机制，我们评估了分流和对照羔羊肺组织中二氢生物蝶呤（BH 2）和四氢生物蝶呤（BH 4）的水平。我们的数据表明，虽然BH 4水平没有变化，但BH 2水平显著增加。最后，我们证明了BH 2的加入增加了纯化的重组eNOS的超氧化物生成。总之，我们的数据表明，肺动脉高压在分流羔羊的发展与氧化应激的增加，不能解释的抗氧化剂的表达或活性的降低。相反，所观察到的氧化应激的增加至少部分是由于BH 2水平升高导致NADPH氧化酶复合物和未偶联eNOS的表达和活性增加。
Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) in lung tissues of Shunt and Control lambs. Our data indicated that although BH4 levels were unchanged, BH2 levels were significantly increased. Finally, we demonstrated that the addition of BH2 produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH2.