Endothelial Cell Dysfunction in Oxidative Stress Models

描述(由申请人提供)：内皮细胞功能障碍是包括糖尿病在内的心血管疾病的主要基础。有证据表明 补充L-精氨酸(L-精氨酸)在逆转中具有治疗作用 内皮功能障碍与心血管疾病的治疗，但其机制 这一效应的影响尚不清楚。因此，我们正在研究氧化的影响 L-精氨酸对内皮细胞转运的损伤及其与内皮的关系 利用糖尿病冠状动脉疾病的实验模型研究糖尿病的功能障碍。 血管系统的正常功能在很大程度上取决于一氧化氮 (NO)血管内皮细胞(EC)产生。然而，在条件下， 与糖尿病等氧化性血管损伤有关， 动脉粥样硬化，高同型半胱氨酸血症，活性氧过量形成 物种可导致内皮功能障碍和NO减少 生物利用度。一氧化氮是由底物L精氨酸合成的一氧化氮合酶产生的。 当L-arg对一氧化氮合酶的可用性有限时，一氧化氮合酶主要作用于0，to 形成超氧化物(O-)，它迅速与NO结合形成过氧亚硝酸根 (ONOO)。ONOO-和02+的形成可导致O-NOS的进一步形成，原因是 氧化BH4(四氢生物蝶呤)，一氧化氮合酶的关键辅助因素。在欧共体， L-精氨酸对一氧化氮合酶的供应主要取决于特定的 传送机，系统y+。我们的数据显示，持续没有接触氧化剂 抑制L-精氨酸的系统转运，降低L-精氨酸的利用度 导致O2的形成这一EC病理被补充 L-阿格。我们假设内皮细胞损伤是由活性氧介导的 物种(ROS)降低了L-精氨酸的转运功能。这降低了L-精氨酸的摄取和 将一氧化氮合酶活性从NO产生转移到O2-产生，导致进一步 L-阿格运输机的妥协。这些有害影响是可以预防的。 补充L-arg。我们的具体目标将检验这些假设，并 进一步表征了L-Arg转运蛋白的调控。目标1.假设： 慢性暴露10ROS可导致L-精氨酸转运体功能障碍。为了测试 在这一假设下，我们将确定长期接触一氧化氮合酶的影响 激动剂、NO供体、02+、ONOO-对人冠状动脉摄取[~3H]L-精氨酸的影响 动脉内皮细胞和B)离体兔心脏灌流的朗宁多夫程序。 目的2.假说--L-精氨酸摄取的减少改变了一氧化氮合酶的活性 生产转O-生产导致L-阿格的进一步妥协 传送器。我们将使用目标1的氧化剂处理方案来关联 基础和一氧化氮合酶激动剂刺激内皮细胞产生NO、O2-和ONOO-的L-Arg 运输活动。目标3：假设：接触氧化剂会改变转运蛋白 蛋白表达、亚细胞分布和/或分子相互作用 伊诺斯。最近的研究表明，L精氨酸对eNOS的主要供应发生在 在小窝内，L-精氨酸转运蛋白CAT1与eNOS相互作用。 因此，氧化剂暴露可能通过改变CAT而抑制L-Arg转运！表达式 水平、亚细胞区隔和/或蛋白质-蛋白质相互作用 和伊诺斯一起。这些系统的相互作用将通过暴露在 HCAEC对上述氧化剂处理的影响及对猫的影响 内皮型一氧化氮合酶的表达、亚细胞分布及分子相互作用 使用免疫沉淀、免疫印迹、亚细胞分级和 共聚焦显微镜。目标4.假设：高血糖/糖尿病导致 内皮功能障碍和降低NO的生物利用度 O2-和ONOO的形成改变了L-Arg转运体的功能， 氧化四氢生物蝶呤，并将eNOS活性从NO转变为02- 制作。这一假设将通过以下实验进行验证：a) 高糖/糖尿病对L-精氨酸转运影响的相关性研究 HCAE中02-和ONOO-对eNOS表达、活性及NO形成的影响 暴露在高糖或控制条件下，并在冠状动脉循环中 从糖尿病兔心脏中分离出来；以及B)确定补充剂 L-精氨酸可有效预防高糖/糖尿病对大鼠血管紧张素转换酶的影响 以上参数。

DESCRIPTION (Provided by applicant): Endothelial cell dysfunction is a primary basis of cardiovascular disease including diabetes mellitus. Evidence suggests that supplemental L-arginine (L-arg) is therapeutically useful in reversing endothelial dysfunction and treating cardiovascular disease, but the mechanism of this effect is unknown. Therefore, we are studying the impact of oxidative injury on endothelial cell transport of L-arg and how it relates to endothelial dysfunction by using experimental models of diabetic coronary artery disease. The normal function of the vascular system depends critically on nitric oxide (NO) production by vascular endothelial cells (EC). However, in conditions associated with oxidative vascular injury such as diabetes mellitus, atherosclerosis, and hyperhomocystememia, excess formation of reactive oxygen species can lead to endothelial dysfunction and reduction in NO bioavailability. NO is produced by NO synthase (NOS) from its substrate L-arg. When L-arg availability to NOS is limiting, NOS acts principally upon 0, to form superoxide (O-), which rapidly combines with NO to form peroxynitrite (ONOO). ONOO- and 02+formation can lead to further formation of O NOS due to oxidation of BH4 (tetrahydrobiopterin), a critical co-factor for NOS. In EC, supply of L-arg to NOS depends mainly on the function of a specific transporter, system y+. Our data show that continued NO oxidant exposure inhibits system y transport of L-arg, reducing availability of L-arg and leading to formation of O2 This EC pathology is reversed with supplemental L-arg. We hypothesize that endothelial cell injury mediated by reactive oxygen species (ROS) reduces L-arg transport function. This reduces L-arg uptake and shifts NOS activity from NO production to O2 - production, leading to further compromise of the L-arg transporter. These deleterious effects can be prevented with supplemental L-arg. Our specific aims will test these hypotheses and further characterize the regulation of L-arg transporter. Aim 1. HYPOTHESIS: Chronic exposure 10 ROS causes dysfunction of the L-arg transporter. To test this hypothesis, we will determine the effects of chronic exposure to NOS agonists, NO donors, 02+-, ONOO- on uptake of [3H]L-arg in A) human coronary artery ECs and B) isolated rabbit hearts perfused by the Langendorff procedure. Aim 2. HYPOTHESIS.- Reduction of L-arg uptake shifts NOS activity from NO production to O - production leading to further compromise of the L-arg transporter. We will use the oxidant treatment protocols of aim 1 to correlate basal and NOS agonist-stimulated EC production of NO, O2 - and ONOO- with L-arg transport activity. Aim 3. HYPOTHESIS: Oxidant exposure alters transporter protein expression subcellular distribution and/or molecular interactions with eNOS. Recent studies indicate the principal supply of L-arg to eNOS occurs within caveolae where the L-arg transporter protein CAT1 interacts with eNOS. Thus, oxidant exposure may inhibit L-arg transport by altering CAT! expression levels, subcellular compartmentalization and/or protein-protein interactions with eNOS. Interactions of these systems will be tested by experiments exposing HCAEC to the above oxidant treatments and determing the effects on CAT expression, subcellular distribution and molecular interactions with eNOS by using immunoprecipitation, immunoblotting, subcellular fractionation and confocal microscopy. Aim 4. HYPOTHESIS: High glucose/ diabetes causes endothelial dysfunction and reduces the bioavailability of NO by increasing formation of O2 - and ONOO which alters function of the L-arg transporter, oxidizes tetrahydrobiopterin, and shifts eNOS activity from NO to 02 - production. This hypothesis will be tested by the following experiments: A) determining the effects of high glucose/diabetes on L-arg transport in relation to eNOS expression and activity and formation of NO of 02 - and ONOO- in HCAEs exposed to high glucose or control conditions and in the coronary circulation isolated from diabetic rabbit hearts; and B) determining whether supplemental L-arg is effective in preventing the effects of high glucose/diabetes on the above parameters.