Reactive nitrogen species and accelerated atherosclerosis

描述（由申请人提供）：有证据表明，来自内皮型一氧化氮合酶（eNOS）的一氧化氮（NO）的活性氮物种（RNS），如过氧亚硝酸盐（ONOO-），在包括糖尿病在内的心血管疾病中很重要。这些氧化剂对“经典”脂溶性抗氧化剂（如维生素E）不敏感，这可能解释了为什么抗氧化治疗在提供持续改善方面是无效的。然而，糖尿病增加RNS的机制以及RNS改变血管功能的机制尚不清楚。我们的初步研究为高血糖和游离脂肪酸（FFA）如何增加RNS及其对细胞功能影响的介导机制建立了新的见解。将培养的人主动脉内皮细胞（HAEC）暴露于临床相关浓度的葡萄糖（20 mM）和FFA（高达0.5 mM）中3天，会增加NO和O2-的产生，并因此降低NO的生物活性，如环状GMP水平降低所示。进一步的证据表明，NO通过与02-反应形成反应产物ONOO-而失活，其与酪氨酸，3-硝基酪氨酸（3-NT）的反应产物水平增加。虽然许多蛋白质的功能可能受到影响，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；在高血糖/FFA患者生长的HAEC中，硝化PGIS水平升高，活性降低。这不仅可以解释为什么糖尿病会降低PGI2水平，也可以解释为什么其前体PGH2会增加，而PGH2会激活血栓素A2受体（称为TP受体，TPr）。我们的初步研究也表明，激活TPr可以调节HAEC中粘附分子的表达和细胞凋亡。此外，TPr拮抗剂或环氧化酶（COX）抑制均可显著降低这些粘附分子的表达和细胞凋亡，提示PGH2激活TPr发生在暴露于高血糖/FFA的HAEC中。因此，我们的中心假设是，糖尿病通过高血糖/高脂血症增加了02-的产生，然后是ONOO'，导致eNOS解耦、PGIS硝化和TPr刺激。这有助于糖尿病血管并发症的发生和发展，因为NO和PGI2的保护作用下调，因为非代谢的PGH2使血小板聚集、动脉粥样硬化积累和血栓形成的平衡发生变化。因此，本研究的目的是：(1)阐明高血糖和FFA增加培养的HAEC中NO和O2-及其反应产物ONOO-的生成以及PGIS的硝化和失活的机制；(2)在高血糖和FFA引起氧化应激增加的情况下，确定TPr刺激对内皮细胞粘附分子表达和凋亡的促进作用；(3)在转基因小鼠和基因敲除小鼠中，确定氧化应激和PGIS失活是否与糖尿病增强的动脉粥样硬化有关。

DESCRIPTION (provided by applicant): There is evidence that reactive nitrogen species (RNS) derived from nitric oxide (NO) of endothelial nitric oxide synthase (eNOS), such as peroxynitrite (ONOO-), are important in cardiovascular diseases including diabetes. These oxidants would not be sensitive to "classical" lipid-soluble antioxidants such as vitamin E, which might explain why antioxidant therapy is ineffective in delivering sustained improvement. However, the mechanisms by which diabetes increases RNS, and those by which RNS modifies vascular functions are poorly understood. Our preliminary studies have established new insights into how hyperglycemia and free fatty acids (FFA) increase RNS and the mechanisms by which its effects on cell function are mediated. Exposure of cultured human aortic endothelial cells (HAEC) to clinically relevant concentrations of glucose (20 mM) and FFA (up to 0.5 mM) for 3 days additively increases the production of both NO and 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 02- to form the reaction products, ONOO-, is found in the increased levels of its reaction product with tyrosine, 3-nitrotyrosine (3-NT). 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 increases and its activity decreases in HAEC grown in hyperglycemia/FFA. This may not only explain why diabetes decreases levels of PGI2, but also why an increase has been noted in its precursor PGH2 which activates upon thromboxane A2 receptor (termed TP receptor, TPr). Our preliminary studies have also shown that activation of TPr can modulate both adhesion molecule expression and apoptosis in HAEC. In addition, either TPr antagonist or inhibition of cyclooxygenase (COX) significantly attenuates both the expression of these adhesion molecules and apoptosis, suggesting activation of TPr by PGH2 occurs in HAEC exposed to hyperglycemia/FFA. Thus, our central hypothesis is that diabetes via hyperglycemia/hyperlipdemia increases the generation of 02- and then ONOO', resulting in eNOS uncoupling, PGIS nitration, and TPr stimulation. This contributes to the initiation and progression of vascular complications in diabetes mellitus because of the down-regulation of protective actions of NO and PGI2 and because the non-metabolized PGH2 tips the balance towards platelet aggregation, atheroma accumulation, and thrombus formation. Thus, the aims of the proposed studies are: (1) To elucidate the mechanism by which hyperglycemia and FFA increases the production of NO and O2-, as well as its reaction product ONOO-, and nitration and inactivation of PGIS in cultured HAEC; (2) To determine the role of TPr stimulation in enhancing endothelial cell adhesion molecules expression and apoptosis under conditions of increased oxidant stress caused by hyperglycemia and FFA; (3) To determine if oxidant stress and PGIS inactivation contribute to the diabetes-enhanced atherogenesis in transgenic and knockout mice.