Oxidative Stress And Antioxidants in Iron Overload

描述（由申请人提供）：异常铁沉积导致肝脏和心脏衰竭，在此之前是组织纤维化。我们发现β受体阻滞剂类似物d-普萘洛尔（d-Pro）在内皮溶酶体中积累，并减少由铁过载引起的细胞和心脏损伤。该建议基于以下假设：铁负载的溶酶体构成了能够诱导体内氧化应激和组织损伤的可释放铁的主要来源。（心脏和肝脏）纤维化，以及增强的心肌对体外施加的缺血/再灌注[I/R]应激的敏感性）;血管紧张素II（Ang II）可促进溶酶体铁蓄积，加重氧化应激、组织纤维化和心肌I/R不耐受;而d-Pro及其主要代谢物4-HO-普萘洛尔（4-OH-Pro）由于其有效的促溶酶体/抗氧化性质而减弱铁介导的纤维化和相关损伤。通过使用大鼠整体模型、离体心脏模型和培养细胞模型，我们提出：1）确定在铁过载期间，在心脏和肝脏氧化应激和纤维化之前组织铁蓄积的程度; 2）研究Ang II如何在体内促进这种发病机制，并评估体内干预的益处（Ang II受体阻断剂、促溶酶体剂和/或金属螯合剂）对组织应激参数和心肌I/R不耐受的影响; 3）研究Ang II介导的铁积累和氧化反应的细胞机制（NADPH氧化酶活化、纤维化活性、蛋白质和脂质氧化），使用体外巨噬细胞、内皮细胞、肝细胞和星状细胞模型;和4）评估Ang II受体阻断剂和d-Pro/4-OH-Pro对铁转运和相关氧化反应的细胞特异性保护。将使用各种复杂的生物物理学（ESR自旋捕获）、免疫化学和分子生物学技术（实时PCR）来评估导致组织氧化应激、纤维化和细胞毒性的事件序列。还将使用gp91 phox敲除小鼠（分包）评估铁过载期间NADPH氧化酶的参与。将使用灵敏的超声心动图技术在小鼠中原位确定铁过载期间心脏功能缺陷的早期指征（分包），并确定上述治疗的影响。拟议的研究可能揭示使用Ang II受体阻断剂和促溶酶体/抗氧化剂作为铁超载患者的辅助治疗的潜在益处。

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.