Oxidants, Eicosanoids and Endothelium in Diabetes

我们的初步研究建立了对高血糖如何增加氧化应激以及 它对细胞功能的影响是通过什么机制来调节的。培养的人内皮细胞的暴露 高糖7-10天会增加NO和超氧阴离子(Oa-)的产生。不是的 通过与02-反应而失活，以形成反应产物过氧亚硝酸根(OONO)，并增加 在细胞中发现其与酪氨酸的反应产物--硝基酪氨酸。尽管许多蛋白质的功能 可能受到影响，我们发现前列环素合成酶(PGIs)在体内被酪氨酸硝化失活 内皮细胞在高糖环境中生长。这可能不仅解释了为什么糖尿病会降低 PGI2，也是为什么激活血栓素A2的PGI2前体PGH2增加的原因 受体(TPR)。TPR活化可促进VCAM-1和ICAM-1的表达及细胞凋亡 人体内皮细胞。黏附分子的表达和细胞凋亡，这两个事件在 动脉粥样硬化的形成也被O2-增强，事实上，暴露在高糖环境中会增强粘附性 通过依赖O2-和TPR的机制进行分子表达和细胞凋亡。事实上，我们已经发现 阻断TPR可抑制载脂蛋白E缺乏的糖尿病致动脉粥样硬化的显著增强 老鼠。我们的总体假设是内皮细胞氧化应激在动脉粥样硬化形成中的作用 作为PGI失活的结果，重要的是通过TPR介导的。此外，在这方面， 计划中，我们发现AMP激酶的刺激物AICAR可以防止内皮细胞的氧化应激 并抑制肿瘤坏死因子-1诱导的血管细胞黏附分子-1表达。我们的目标是：1) 确定葡萄糖和脂肪酸升高增加NO和02-的产生的机制 导致酪氨酸硝化和PGI合酶失活。AMP激酶在氧化剂调节中的作用 通过PKC的压力将被研究。2)确定二十烷类化合物对TPR的刺激作用 内皮细胞氧化应激和PGI合成酶失活导致白细胞粘附性增加和 由高糖和脂肪酸引起的细胞凋亡，以及3)确定氧化应激，PGIs失活， TPR可对抗转基因小鼠糖尿病致动脉粥样硬化的增加。激活 AMP激酶作为减少氧化应激的策略将被探索。

Our preliminary studies establish new insights into how elevated glucose increases oxidant stress and the mechanisms by which its effects on cell function are mediated. Exposure of cultured human endothelial cells to elevated glucose for 7-10 days increases the production of both NO and superoxide anion (Oa-). NO is inactivated by reacting with 02- to form the reaction product, peroxynitrite (OONO), and increased levels of its reaction product with tyrosine, nitrotyrosine, is found in the cells. Although the fimction of many proteins may be affected, we have found that prostacyclin synthase (PGIS) is inactivated by tyrosine nitration in endothelial cells grown in elevated glucose. This may not only explain why diabetes decreases levels of PGI2, but also why increases have been noted in the PGI2 precursor, PGH2, which activates thromboxane A2 receptors (TPr). Activation of TPr can increase VCAM-1 and ICAM-1 expression as well as apoptosis of human endothelial cells. Expression of adhesion molecules and apoptosis, two events important in atherogenesis, are also enhanced by O2-, and indeed, exposure to elevated glucose enhances adhesion molecule expression and apoptosis by mechanisms which depend on O2- and TPr. Indeed, we have found that blockade of TPr inhibits the dramatic enhancement by diabetes ofatherogenesis in the Apo E deficient mouse. Our overall hypothesis is that the effects of oxidant stress in the endothelium in atherogenesis are importantly mediated via TPr as a result of inactivation of PGIS. Furthermore, in the context of this program, we have found that AICAR, a stimulator of AMP kinase, prevents the oxidant stress in endothelial cells exposed to elevated glucose, and inhibits VCAM-1 expression induced by TNF_. Our aims are: 1) To determine the mechanism by which elevated glucose and fatty acids increases production of NO and 02- and causes tyrosine nitration and inactivation ofPGI synthase. The role of AMP kinase in regulating oxidant stress via PKC will be studied. 2) To determine the role of TPr stimulation by eicosanoid products due to endothelial cell oxidant stress and PGI synthase inactivation in causing the increased leukocyte adhesion and apoptosis caused by high glucose and fatty acids, and 3) to determine if oxidant stress, PGIS inactivation, and TPr contr_ute to the increased atherogenesis caused by diabetes in transgenie mouse models. Activation of AMP kinase as a strategy for reducing oxidant stress will be explored.