IRON-MEDIATED CARDIOVASCULAR INJURY

铁介导的心血管损伤

• 批准号: 2332550
• 负责人: LAWRENCE HORWITZ
• 金额: $ 24.09万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332550
• 关键词: Adenoviridae; ammonium chloride; chelating agents; chloroquine; ferritin; free radical oxygen; gas chromatography; gene targeting; heart cell; hydroxyl radical; iron; iron metabolism; laboratory rabbit; laboratory rat; lactoferrin; mass spectrometry; myocardial ischemia /hypoxia; oxidative stress; recombinant virus; reperfusion; siderophores; tissue /cell culture; transferrin; transferrin receptor

We propose that substantial reductions in the morbidity and mortality of acute myocardial infarction can be achieved by new approaches aimed at preventing the iron-mediated damage that occurs during reperfusion of ischemic myocardium. Our major hypothesis is: Production of reactive oxygen species during reperfusion causes injury due to .OH production or other processes dependent upon availability of iron, which is determined by transferrin and ferritin regulation of the cellular labile iron pool. We will investigate, using molecular biological methods in cultured cardiac myocytes, the cellular regulation of free iron. We will then ascertain the degree to which free iron is involved in oxidant injury in cultured myocytes, and apply our findings to potential therapeutic interventions in isolated, perfused rabbit hearts. In AIM 1, we will infect cultured adult rat cardiac myocytes with recombinant adenoviruses encoding constitutively expressed, unregulated, or inducible, iron- regulated, human transferrin receptors. We will examine the effect of infection with these adenoviruses on iron-transferrin uptake and cellular free iron and ferritin levels. Under conditions simulating oxidant exposure during reperfusion, we will measure cell injury, lipid peroxidation and glutathione oxidation, all of which we postulate will be increased in the genetically altered myocytes with unregulated transferrin receptors. In AIM 2 we will study a group of unique, newly discovered iron chelators, the exochelins of Mycobacterium tuberculosis, which prevent .OH production and are both water and lipid soluble. These agents enter cells more rapidly than other chelators and have a very high binding affinity for iron. We anticipate that the exochelins will reduce intracellular iron levels and susceptibility to oxidant injury. In AIM 3 we will examine in cultured myocytes and isolated perfused rabbit hearts several measures to reduce intracellular iron. In cultured myocytes we will study a receptor- dependent iron chelator, lactoferrin, and the effect of agents (chloroquine and ammonium chloride) that increase pH in endocytic vesicles and lysosomes, thereby inhibiting release of iron from transferrin and ferritin. In conjunction with these studies we will employ a new highly sensitive assay for .OH, which employs gas chromatography and mass spectrometry detection of salicylate isomers of this radical. Further studies of iron-directed potential therapies will measure ventricular function, myocardial enzyme release and lipid peroxidation in isolated hearts exposed to hypoxia and reoxygenation, using the chelators and other methods for reducing intracellular free iron which were previously examined in the experiments with cultured cardiac myocytes.

我们建议，大幅度降低发病率和死亡率， 急性心肌梗死可以通过新的方法来实现， 防止再灌注期间发生的铁介导的损伤， 缺血心肌我们的主要假设是： 再灌注过程中的氧物质由于.OH的产生而引起损伤， 其他工艺取决于铁的可用性， 通过转铁蛋白和铁蛋白调节细胞不稳定铁池。 我们将利用分子生物学方法， 心肌细胞，游离铁的细胞调节。 然后我们将 确定游离铁参与氧化损伤的程度， 培养的心肌细胞，并将我们的发现应用于潜在的治疗 在离体灌注的兔心脏中进行干预。在AIM 1中，我们将 重组腺病毒感染培养成年大鼠心肌细胞 编码组成型表达的、不受调节的或诱导的铁- 受调节的人转铁蛋白受体。我们将研究 这些腺病毒感染对铁-转铁蛋白摄取和细胞 游离铁和铁蛋白水平。在模拟氧化剂的条件下 暴露在再灌注期间，我们将测量细胞损伤，脂质 过氧化和谷胱甘肽氧化，我们假设所有这些都会 在转铁蛋白不受调节的遗传改变的肌细胞中增加 受体。在AIM 2中，我们将研究一组新发现的独特铁 螯合剂，结核分枝杆菌的外螯合素，其防止。 并且是水溶性和脂溶性的。这些物质进入细胞 比其他螯合剂更快，并具有非常高的结合亲和力 铁。我们预期外螯素将减少细胞内铁 水平和对氧化损伤的敏感性。在AIM 3中，我们将检查 培养的心肌细胞和离体灌流的兔心脏几种措施， 减少细胞内铁。在培养的心肌细胞中我们将研究一种受体- 依赖性铁螯合剂，乳铁蛋白，以及药物的作用 （氯喹和氯化铵）增加内吞囊泡的pH值 和溶酶体，从而抑制铁从转铁蛋白的释放， 铁蛋白结合这些研究，我们将采用一种新的高度 采用气相色谱法和质谱法， 光谱法检测该自由基的水杨酸盐异构体。进一步 铁导向电位疗法的研究将测量心室 心肌酶释放和脂质过氧化反应 心脏暴露于缺氧和复氧，使用螯合剂和其他 用于减少细胞内游离铁的方法， 在培养的心肌细胞的实验中检查。