Endothelial Cell Dysfunction in Oxidative Stress Models

描述（由申请方提供）：内皮细胞功能障碍是原发性 糖尿病等心血管疾病的基础。证据表明 补充L-精氨酸（L-arg）在治疗上可用于逆转 内皮功能障碍和治疗心血管疾病，但机制 这种影响是未知的。因此，我们正在研究氧化 L-精氨酸对内皮细胞转运损伤及其与内皮细胞的关系 通过使用糖尿病冠状动脉疾病的实验模型， 血管系统的正常功能主要依赖于一氧化氮 (NO)由血管内皮细胞（EC）产生。然而，在条件 与氧化性血管损伤如糖尿病有关， 动脉粥样硬化和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NO由NO合成酶（NOS）从其底物L-arg产生。 当NOS的L-arg可用性受限时，NOS主要作用于0， 形成超氧化物（O-），迅速与NO结合形成过氧亚硝酸盐 （ONOO）. ONOO-和O2+的形成可以导致ONOS的进一步形成，这是由于 氧化BH 4（四氢生物蝶呤），一个关键的辅因子NOS。在欧共体， L-arg向NOS的供应主要取决于一个特定的 传送器Y+系统我们的数据表明，持续的NO氧化剂暴露 抑制L-arg的系统转运，降低L-arg的可用性， 导致O2的形成。 L-精氨酸我们推测活性氧介导的内皮细胞损伤 活性氧（ROS）降低L-arg转运功能。这减少了L-arg摄取， 将NOS活性从NO产生转移到O2产生，导致进一步的 L-arg转运蛋白的受损这些有害影响是可以预防的 补充L-精氨酸我们的具体目标将检验这些假设， 进一步表征L-arg转运蛋白的调节。目标1.假设： 慢性暴露10 ROS导致L-arg转运蛋白功能障碍。测试 根据这一假设，我们将确定长期暴露于NOS的影响 激动剂、NO供体、O2 +-、ONOO-对A）人冠状动脉中[3 H]L-arg摄取的影响 动脉EC和B）通过Langendorff程序灌注的离体兔心脏。 目标2.假设。L-arg摄取的减少使NOS活性从NO 生产到O -生产导致L-arg的进一步损害 传送器我们将使用aim 1的氧化剂处理方案来关联 基础和NOS激动剂用L-arg刺激EC产生NO、O2 -和ONOO- 运输活动。目标3.假设：氧化剂暴露改变转运蛋白 蛋白质表达、亚细胞分布和/或分子相互作用 eNOS。最近的研究表明，eNOS的L-arg主要供应发生在 在L-arg转运蛋白CAT 1与eNOS相互作用的小窝内。 因此，氧化剂暴露可通过改变CAT！表达 水平、亚细胞区室化和/或蛋白质-蛋白质相互作用 关于eNOS这些系统的相互作用将通过实验进行测试， HCAEC对上述氧化剂处理的影响，并测定其对CAT的影响 表达，亚细胞分布和与eNOS的分子相互作用， 使用免疫沉淀、免疫印迹、亚细胞分级分离和 共聚焦显微镜目标4。假设：高血糖/糖尿病原因 内皮功能障碍，并通过增加NO的生物利用度来降低NO的生物利用度。 O2 -和ONOO的形成改变了L-arg转运蛋白的功能， 氧化四氢生物蝶呤，并将eNOS活性从NO转移到O2- 生产这一假设将通过以下实验进行检验： 确定高糖/糖尿病对L-arg转运的影响， 对内皮型一氧化氮合酶的表达和活性及一氧化氮的形成有影响 暴露于高葡萄糖或对照条件和冠状循环中 从糖尿病兔心脏分离;和B）确定是否补充 L-arg可有效预防高糖/糖尿病对 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.