Normal vascular development in mice deficient in endothelial NO synthase: possible role of neuronal NO synthase.
Normal vascular development in mice deficient in endothelial NO synthase: possible role of neuronal NO synthase.
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发表时间：
2003-10
期刊：
影响因子：
2.2
通讯作者：
M. Al-Shabrawey;A. El-Remessy;X. Gu;Steven S. Brooks;M. S. Hamed;Paul L Huang;R. Caldwell
中科院分区：
文献类型：
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作者：
M. Al-Shabrawey;A. El-Remessy;X. Gu;Steven S. Brooks;M. S. Hamed;Paul L Huang;R. Caldwell
目的一氧化氮合酶（NOS）在氧诱导视网膜病变过程中产生的一氧化氮参与了血管损伤和视网膜新生血管的形成。然而，NOS在正常视网膜血管发育和生长中的作用尚未研究。这些实验的目的是表征与血管发育有关的NOS的表达，并确定删除内皮NOS（eNOS）对这一过程的影响。方法采用形态计量学和生物化学相结合的方法，对150只1 d ~ 6月龄的eNOS+/+和eNOS-/-小鼠视网膜血管发育进行分析。在视网膜组织切片和用荧光素缀合的Griffonia simplicifolia凝集素标记的整装制剂中分析血管发育的模式。血管密度和动脉直径的分析与凝集素标记的整体安装使用计算机辅助形态计量学。通过使用Greiss反应测量硝酸盐/亚硝酸盐积累的视网膜水平来定量NO产生。用特异性NOS抗体的Western印迹技术来评估NOS蛋白表达水平的差异。采用nNOS免疫细胞化学和NADPH黄递酶组织化学方法观察nNOS在视网膜的分布。结果eNOS-/-小鼠视网膜血管发育的速率和模式与野生型对照小鼠相当。血管密度的测量显示两个品系之间没有显著差异。eNOS-/-视网膜中的NO产生量也与eNOS+/+视网膜中的相当。对eNOS+/+和eNOS-/-小鼠中nNOS表达的分析显示，两种品系中总nNOS蛋白水平相似。诱导型NOS在两种菌株中均未检测到。nNOS分布的研究表明，在eNOS-/-视网膜的深毛细血管丛的强烈标记。这在野生型视网膜中没有观察到。在eNOS-/-小鼠中显示NADPH-黄递酶活性的神经元细胞的数量也显著增加。结论：在没有eNOS的情况下，视网膜血管的发育正常。在eNOS-/-视网膜中观察到类似水平的NO产生、nNOS的血管周围再分布和NADPH-黄递酶反应性神经元的数量增加，表明血管相关的nNOS活性的增加补偿了发育中的突变视网膜中的eNOS缺乏。
PURPOSE Nitric oxide formation by nitric oxide synthase (NOS) has been implicated in vascular injury and retinal neovascularization during oxygen-induced retinopathy. However, the role of NOS in normal retinal vascular development and growth has not been studied. The purpose of these experiments was to characterize the expression of NOS in relation to vascular development and to determine the effect of deleting endothelial NOS (eNOS) on this process. METHODS Retinal vascular development was analyzed in 150 eNOS+/+ and eNOS-/- mice ranging from 1 day to 6 months old by using a combination of morphometric and biochemical approaches. The pattern of vascular development was analyzed in retinal tissue sections and whole-mount preparations labeled with fluorescein-conjugated Griffonia simplicifolia lectin. Analysis of vascular density and arterial diameter were performed with the lectin-labeled whole-mounts using computer-assisted morphometry. NO production was quantified by measuring retinal levels of nitrate/nitrite accumulation using the Greiss reaction. Western blotting techniques with isoform-specific NOS antibodies were used to evaluate differences in levels of NOS protein expression. Retinal distribution of nNOS was characterized using nNOS immunocytochemistry and NADPH diaphorase histochemistry. RESULTS These analyses showed that the rate and pattern of retinal vascular development in eNOS-/- mice were comparable with those in wild-type control mice. Measurement of vascular density showed no significant differences between the two strains. The amount of NO production in the eNOS-/- retina was also equivalent to that in the eNOS+/+ retina. Analysis of nNOS expression within the eNOS+/+ and eNOS-/- mice showed similar levels of total nNOS protein in the two strains. Inducible NOS was not detected in either strain. Studies of nNOS distribution showed intense labeling of the deep capillary plexus in the eNOS-/- retina. This was not seen in the wild-type retinas. The number of neuronal cells showing NADPH-diaphorase activity was also significantly increased in the eNOS-/- mice. CONCLUSIONS Development of the retinal vasculature occurs normally without eNOS. The observations of similar levels of NO production, perivascular redistribution of nNOS and increased numbers of NADPH-diaphorase reactive neurons in the eNOS-/- retinas suggest that increases in vascular-associated nNOS activity compensate for the eNOS deficiency in the developing mutant retina.