Oxidative Stress And Antioxidants in Iron Overload

描述(由申请人提供)：异常的铁沉积导致肝脏和心脏衰竭，在此之前会出现组织纤维化。我们发现，β-受体阻滞剂类似物d-心得安(d-Pro)积聚在内皮溶酶体中，可以减轻铁超载引起的细胞和心脏损伤。这一建议基于下列假设：负载铁的溶酶体构成可释放铁的主要来源，可诱导体内氧化应激和组织(心脏和肝脏)纤维化，以及增加心肌对体外缺血/再灌注[I/R]应激的敏感性)；血管紧张素II(Ang II)可促进溶酶体铁积聚并加剧氧化应激、组织纤维化和心肌耐受；d-Pro及其主要代谢物4-HO-心得安(4-HO-Pro)因其强大的溶酶体/抗氧化特性而延缓铁介导的纤维化和相关损伤。通过建立整体大鼠、离体心和培养细胞模型，我们建议：1)确定铁超载时心脏和肝脏氧化应激和纤维化之前组织铁积累的程度；2)研究Ang II如何在体内促进这一发病机制，并评估体内干预(Ang II受体阻滞剂、溶酶促进剂和/或金属螯合)对组织应激参数和心肌I/R不耐受的益处；3)利用体外巨噬细胞、内皮细胞、肝细胞和星形细胞模型，研究Ang II介导的铁积累和氧化反应(NADPH氧化酶激活、纤维化活性、蛋白质和脂质氧化)的细胞机制；4)评价Ang II受体阻断剂和d-Pro/4-OH-Pro对铁转运及相关氧化反应的细胞特异性保护作用。各种复杂的生物物理(ESR自旋捕获)、免疫化学和分子生物学技术(实时PCR)将被用来评估导致组织氧化应激、纤维化形成和细胞毒性的一系列事件。也将使用gp91Phox基因敲除小鼠(分包合同)来评估NADPH氧化酶在铁超载过程中的参与。铁负荷期间心脏功能缺陷的早期指征将使用灵敏的超声心动学技术在小鼠身上原位确定(分包)，并确定上述治疗的影响(S)。拟议的研究可能揭示使用血管紧张素Ⅱ受体阻滞剂和溶酶促性剂/抗氧化剂作为铁负荷过高患者的辅助治疗的潜在好处。

DESCRIPTION (provided by applicant): Abnormal iron deposition causes liver and cardiac failure that is preceded by tissue fibrosis. We showed that the beta-blocker analog, d-propranolol (d-Pro), accumulates in endothelial lysosomes and reduces both cellular and cardiac injury caused by iron overload. This proposal is based upon the following hypotheses: Iron-loaded lysosomes constitute a major source of releasable iron capable of inducing in vivo oxidative stress and tissue (cardiac and hepatic) fibrosis, as well as enhanced myocardial susceptibility to imposed ischemia / reperfusion [I/R] stress in vitro); Angiotensin II (Ang II) may promote lysosomal iron accumulation and exacerbate oxidative stress, tissue fibrosis and myocardial intolerance to I/R; and d-Pro and its major metabolite, 4-HO-propranolol (4-OH-Pro), attenuate iron-mediated fibrosis and associated injury as a result of their potent lysosomotropic / antioxidant properties. By using both the whole rat, isolated heart and cultured cell models, we propose to: 1) Determine the extent to which tissue iron accumulation occurs prior to cardiac and hepatic oxidative stress and fibrosis during iron overload; 2) Study how Ang II facilitates this pathogenesis in vivo and assess the benefits of in vivo intervention (Ang II receptor blockade, lysosomotropic agents and/or metal chelation) on tissue stress parameters and myocardial intolerance to I/R; 3) Investigate the cellular mechanisms underlying Ang ll-mediated iron accumulation and oxidative responses (NADPH oxidase activation, fibrogenic activity, protein and lipid oxidation) using in vitro macrophage, endothelial cell, hepatocyte, and stellate cell models; and 4) Assess cell-specific protection of Ang II receptor blockade and d-Pro /4-OH-Pro on iron transport and associated oxidative responses. Various sophisticated biophysical (ESR spin trapping), immunochemical and molecular biology techniques (real-time PCR) will be used to assess the sequence of events leading to tissue oxidative stress, fibrogenesis and cellular toxicity. The involvement of NADPH oxidase during iron overload will also be assessed using gp91 phox knockout mice (Subcontract). Early indications of cardiac functional defects during iron overload will be determined in situ using sensitive echocardiologic techniques in the mouse (Subcontract), and the impact of the above treatment(s) determined. The proposed studies may reveal potential benefits of using Ang II receptor blockade and lysosomotropic/antioxidant agents as adjunct therapy for iron-overloaded patients.