IRON-MEDIATED CARDIOVASCULAR INJURY

我们建议大幅降低儿童的发病率和死亡率。 急性心肌梗死可通过针对以下目标的新方法实现 预防再灌流期间发生的铁介导的损伤 缺血心肌。我们的主要假设是：反应性的产生 再灌流过程中的氧物种由于产生.OH或 其他过程取决于铁的可获得性，这是确定的 通过转铁蛋白和铁蛋白调节细胞内不稳定的铁库。 我们将利用分子生物学方法对培养的 心肌细胞，细胞对游离铁的调节。到时候我们会的 确定游离铁在氧化损伤中的参与程度 培养心肌细胞，并将我们的发现应用于潜在的治疗 对隔离、灌流的兔心进行干预。在目标1中，我们将 重组腺病毒感染培养的成年大鼠心肌细胞 编码结构性表达的、不受调控的或可诱导的铁- 受调节的人转铁蛋白受体。我们将研究以下方面的影响 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 在再灌流期间暴露，我们将测量细胞损伤，血脂 过氧化和谷胱甘肽氧化，我们假设所有这些都将是 未调控转铁蛋白基因改变的心肌细胞中含量增加 感受器。在目标2中，我们将研究一组独特的、新发现的铁 螯合剂，结核分枝杆菌的胞外凝集素，可以预防。 可生产，可溶于水和脂类。这些代理人进入细胞 比其他螯合剂更快，并具有非常高的结合亲和力 用来换铁。我们预计胞外蛋白会减少细胞内的铁。 氧化损伤的水平和敏感性。在AIM 3中，我们将在 培养的心肌细胞和分离的灌流兔心的几项措施 减少细胞内的铁。在培养的心肌细胞中，我们将研究一种受体- 依从性铁络合剂、乳铁蛋白及其制剂的作用 (氯喹和氯化铵)可增加内吞囊泡的pH值 和溶酶体，从而抑制铁从转铁蛋白和 铁蛋白。结合这些研究，我们将采用一种新的高度 使用气相色谱和质谱法对.oh进行灵敏的测定 该自由基水杨酸异构体的光谱检测。进一步 铁定向电势疗法的研究将测量脑室 离体心功能、心肌酶释放和脂质过氧化 心脏暴露在低氧和复氧中，使用螯合剂和其他 降低细胞内游离铁的方法 在与培养的心肌细胞的实验中进行了检验。

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.