Oxidants, eicosanoids, and endothelium in diabetes

描述(由申请人提供)：氧化应激被广泛认为在糖尿病环境中暴露于高血糖和高脂血症的细胞中发挥功能变化的作用，并被认为是加速糖尿病动脉粥样硬化的机制。糖尿病增加氧化应激的机制，以及氧化应激改变内皮功能的机制还知之甚少。我们的初步研究对高血糖如何增加氧化应激以及其对细胞功能的影响的调节机制建立了新的见解。培养的人内皮细胞暴露于高糖7-10天会增加NO和超氧阴离子(O2-)的产生，从而降低NO的生物活性，表现为环状GMP水平的降低。进一步的证据表明，NO通过与O2-反应生成反应产物过氧亚硝酸根(00N0-)，在其与细胞中发现的酪氨酸3-o-硝基酪氨酸的反应产物中发现了增加的水平。虽然许多蛋白质的功能可能会受到影响，但我们发现前列环素合成酶(PGIs)特别容易受到酪氨酸硝化的影响；在高糖环境中生长的内皮细胞中，硝化的PGIs水平增加，其活性降低。这可能不仅解释了为什么糖尿病降低了PGI2的水平，而且也解释了为什么它的前体PGH2增加了，它激活了血栓素A2受体(TPR)。我们的研究表明，TPR的激活可以调节人内皮细胞ICAM-1和VCAM-L的表达。黏附分子的表达被02-增强，事实上，暴露在高葡萄糖环境中会增强黏附分子的表达。因此，高糖诱导的氧化应激可能调节PGIs的活性和TPR的刺激，从而调节黏附分子的表达。事实上，我们已经发现，阻断TPR可以抑制载脂蛋白E缺陷小鼠的动脉粥样硬化，在这种模型中，糖尿病促进了动脉粥样硬化的形成。有三个具体目的：1)确定高糖和脂肪酸增加NO和02的产生并导致酪氨酸硝化和PGI合酶失活的机制；2)确定内皮细胞氧化应激和PGI合酶失活引起的二十烷类产物对TPR的刺激在导致高糖和脂肪酸引起的白细胞黏附和凋亡增加中的作用；3)在转基因小鼠模型中确定氧化应激和PGI合酶失活是否导致糖尿病引起的动脉粥样硬化加剧。

DESCRIPTION (provided by applicant): Oxidant stress is widely recognized to play a role in functional alterations that develop in cells exposed to hyperglycemia and hyperlipidemia in the diabetic milieu, and has been implicated as a mechanism that accelerates atherosclerosis in diabetes. The mechanisms by which diabetes increases oxidant stress, and those by which oxidant stress modifies endothelial function are poorly understood. 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 (O2-), and consequently decreases the bioactivity of NO as indicated by decreased levels of cyclic GMP. Further evidence that NO is inactivated by reacting with O2- to form the reaction product, peroxynitrite (00N0-) is found in the increased levels of its reaction product with tyrosine, 3-o-nitrotyrosine, found in the cells. While the function of many proteins may be affected, we have found that prostacyclin synthase (PGIS) is particularly susceptible to tyrosine nitration; the levels of nitrated PGIS increase and its activity decreases 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 its precursor PGH2 that activates thromboxane A2 receptors (TPr). Our studies have shown that activation of TPr can modulate ICAM-1 and VCAM-l expression in human endothelial cells. The expression of adhesion molecules is enhanced by 02-, and indeed, exposure to elevated glucose enhances adhesion molecule expression. Thus, oxidant stress induced by elevated glucose may modulate the activity of PGIS and stimulation of TPr, thereby modulating adhesion molecule expression. Indeed, we have found that blockade of TPr inhibits atherosclerosis in the Apo E deficient mouse, a model in which diabetes enhances atherogenesis. There are three specific aims: 1) to determine the mechanism by which elevated glucose and fatty acids increases production of NO and 02- and causes tyrosine nitration and inactivation of PGI synthase, 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 and PGI synthase inactivation contributes to the increased atherogenesis caused by diabetes in transgenic mouse models.