Reactive nitrogen species & accelerated atherosclerosis

描述（由申请人提供）：有证据表明，源自内皮型一氧化氮合酶（eNOS）的一氧化氮（NO）的活性氮物质（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剂治疗在提供持续改善方面无效。然而，糖尿病增加RNS的机制以及RNS改变血管功能的机制知之甚少。我们的初步研究已经建立了新的见解，高血糖症和游离脂肪酸（FFA）如何增加RNS及其对细胞功能的影响是介导的机制。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）3天，增加了NO和O2-的产生，因此，降低了NO的生物活性，如环GMP水平降低所示。NO通过与O2-反应形成反应产物ONOO-而失活的进一步证据见于其与酪氨酸的反应产物3-硝基酪氨酸（3-NT）的水平增加。虽然许多蛋白质的功能可能会受到影响，我们已经发现，前列环素合酶（PGIS）是特别容易受到酪氨酸硝化;硝化PGIS的水平增加，其活性降低在高血糖症/FFA中生长的HAEC。这不仅可以解释为什么糖尿病会降低PGI 2的水平，而且还可以解释为什么其前体PGH 2会增加，该前体PGH 2会激活血栓烷A2受体（称为TP受体，TPr）。我们的初步研究还表明，激活TPr可以调节HAEC中粘附分子的表达和凋亡。此外，无论是TPr拮抗剂或抑制环氧合酶（考克斯）显着减弱这些粘附分子的表达和细胞凋亡，表明PGH 2激活TPr发生在HAEC暴露于高血糖症/FFA。因此，我们的中心假设是糖尿病通过高血糖症/高血脂症增加了O2-和ONOO '的产生，导致eNOS解偶联、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PGI 2的保护作用下调，并且因为未代谢的PGH 2使平衡倾向于血小板聚集、动脉粥样化积累和血栓形成。（1）阐明高血糖和游离脂肪酸增加HAEC NO和O2-及其反应产物ONOO-的产生、PGIS的硝化和失活的机制;（二）确定TPr刺激在高血糖引起的氧化应激增加条件下增强内皮细胞粘附分子表达和凋亡中的作用FFA;（3）研究氧化应激和PGIS失活是否参与了糖尿病转基因和基因敲除小鼠动脉粥样硬化的形成。

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.