Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy
Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy
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DOI：
10.1167/iovs.07-0112
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发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Forrester, John V.
中科院分区：
文献类型：
--
作者：
Leal, Ermelindo C.;Manivannan, Ayyakkannu;Forrester, John V.
目的.一氧化氮（NO）参与糖尿病视网膜病变（DR）早期白细胞淤滞和血视网膜屏障（BRB）破坏，但目前尚不清楚NO合酶（NOS）亚型主要参与。在这项研究中，作者的目的是阐明参与组成型（eNOS，nNOS）和诱导型NOS（iNOS）亚型和NO介导的白细胞停滞和BRB分解的机制。用链脲佐菌素诱导糖尿病2周。用NOS抑制剂N-G-硝基-L-精氨酸甲酯（L-NAME）处理小鼠，其显示对组成型同种型的偏好超过iNOS。用伊文思蓝评估血管渗漏。白细胞滞留在平板视网膜中用共聚焦显微镜定量，在体内用扫描激光检眼镜定量，在体外用视网膜内皮细胞系定量。ICAM-1、occludin和ZO-1水平通过Western印迹、流式细胞术或免疫组织化学进行评估。硝基酪氨酸含量通过电化学方法进行评估。糖尿病使视网膜血管内白细胞淤滞和BRB通透性增加，而L-NAME可使其减少.在糖尿病iNOS敲除小鼠中观察到类似的效果。在糖尿病小鼠视网膜，ICAM-1蛋白水平增加，而紧密连接蛋白，occludin和ZO- 1的免疫反应性下降，与所有NOS亚型的蛋白水平增加。这些作用被L-NAME阻止，在糖尿病iNOS基因敲除小鼠中也是如此.高糖和亚硝化/氧化应激也增加了ICAM-1上调引起的白细胞停滞。这些结果表明，iNOS亚型在白细胞停滞和BRB崩溃中起着主导作用。其机制涉及ICAM-1上调和紧密连接蛋白下调。
PURPOSE. Nitric oxide ( NO) is involved in leukostasis and blood-retinal barrier ( BRB) breakdown in the early stages of diabetic retinopathy ( DR), but it is unclear which NO synthase ( NOS) isoforms are primarily involved. In this study, the authors aimed to clarify the involvement of constitutive ( eNOS, nNOS) and inducible NOS ( iNOS) isoforms and the mechanisms underlying NO- mediated leukostasis and BRB breakdown.METHODS. Diabetes was induced with streptozotocin for 2 weeks. Mice were treated with a NOS inhibitor, N-G- nitro- L-arginine methyl ester ( L- NAME), which shows a preference for constitutive isoforms over iNOS. Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat- mounted retinas with confocal microscopy, in vivo with a scanning laser ophthalmoscope, and in vitro in a retinal endothelial cell line. ICAM-1, occludin, and ZO-1 levels were assessed by Western blot, flow cytometry, or immunohistochemistry. Nitrotyrosine content was assessed by immunohistochemistry.RESULTS. Diabetes increased leukostasis within retinal vessels and BRB permeability, which were reduced by L- NAME. Similar effects were observed in diabetic iNOS knockout mice. In diabetic mouse retinas, ICAM-1 protein levels increased, whereas the immunoreactivity of tight junction proteins, occludin and ZO- 1 decreased, in correlation with increased protein levels of all NOS isoforms. Those effects were prevented by L- NAME and also in diabetic iNOS knockout mice. High glucose and nitrosative/ oxidative stress also increased leukostasis caused by ICAM-1 upregulation.CONCLUSIONS. These results indicate that the iNOS isoform plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction protein downregulation.