Peroxide, NO and Iron Signaling in Endothelial Damage

描述(由申请人提供)：长期目标：这项建议的长期目标是揭示氧化剂诱导的铁信号机制在内皮细胞凋亡中的作用。新近的研究表明，活性氧和氮(ROS/RNS)引起的细胞氧化损伤受细胞铁稳态的重要控制。假设：需要检验的一般假设是：(I)过氧化氢(如过氧化氢、脂质过氧化氢)诱导的内皮细胞氧化损伤和凋亡是通过转铁蛋白受体(TFR)依赖的转铁蛋白(Tf)-铁摄取而介导的，TFR是氧化剂诱导的内皮细胞凋亡的有效守门人和调节器，(Ii)一氧化氮(NO)和抗氧化剂通过抑制铁信号机制来减轻过氧化诱导的内皮损伤。具体目标：首先，我们将研究细胞外和细胞内过氧化氢对内皮铁信号转导和细胞凋亡的影响。接下来，我们将研究细胞通透性的酯酶特异性NO供体和NO合成酶抑制剂在过氧化氢诱导铁信号转导和细胞凋亡中的作用。这里的目标是确定铁、氧化/亚硝化应激和过氧化氢诱导的内皮毒性之间的联系。最后，我们将研究补充抗氧化剂(酶)对过氧化氢诱导的细胞内氧化应激、铁信号转导和细胞凋亡的影响。方法：采用牛主动脉内皮细胞(BAEC)和人主动脉内皮细胞(HAEC)。将测量以下氧化还原参数：GSH和过氧化脂质；乌头酸酶和铁调节蛋白活性；TFR表达，55Fe摄取；caspase信号转导和细胞凋亡。超氧阴离子和羟基自由基将通过荧光和新的自旋捕获(免疫分析)技术来测定。意义：内皮细胞损伤是许多心血管疾病的早期氧化损伤。一些临床和基础研究表明，氧化剂诱导的铁信号在血管氧化损伤中发挥作用。新颖性：这项提议对过氧化氢诱导的铁信号在内皮细胞凋亡中的作用以及一氧化氮和抗氧化剂在减轻这些影响中的作用提出了新的见解。

DESCRIPTION (provided by applicant): Long-term goal: The long-term goal of this proposal is to unravel the role of oxidant-induced iron signaling mechanism in endothelial cell apoptosis. Emerging studies indicate that the cellular oxidative damage caused by reactive oxygen and nitrogen species (ROS/RNS) is critically controlled by cellular iron homeostasis. Hypothesis: The general hypotheses to be tested are that (i) peroxide (e.g., H2O2, lipid hydroperoxide)-induced endothelial oxidative damage and apoptosis are mediated by transferrin receptor (TfR)-dependent uptake of transferrin (Tf)-iron and that the TfR is an effective "gatekeeper" and modulator of oxidant-induced apoptosis in endothelial cells, and (ii) nitric oxide (NO) and antioxidants mitigate peroxide-induced endothelial damage by inhibiting iron signaling mechanism. Specific aims: First, we will investigate the effect of extracellular and intracellular hydroperoxides on endothelial iron signaling and apoptosis. Next, we will investigate the effect of cell-permeable esterase-specific NO donors and NO synthase inhibitors on peroxide-induced iron signaling and apoptosis. The objective here will be to establish the link between iron, oxidative/nitrosative stress, and peroxide-induced endothelial toxicity. Finally, we will investigate the effect of antioxidant (enzyme) supplementation on peroxide-induced intracellular oxidative stress, iron signaling, and apoptosis. Methods: We will use the bovine aortic endothelial cells (BAEC) and human aortic endothelial cells (HAEC). The following redox- parameters will be measured: GSH and lipid peroxides; aconitase and iron-regulatory protein activities; TfR expression, 55Fe uptake; caspase signaling, and apoptosis. Superoxide and hydroxyl radicals will be determined by fluorescence and novel spin-trapping (immunoassay) techniques. Significance: Endothelial cell injury is an early oxidative insult in many cardiovascular diseases. Several clinical and basic research studies implicated a role for oxidant-induced iron signaling in vascular oxidative damage. Novelty: This proposal presents a new insight on peroxide-induced iron signaling in endothelial apoptosis and on the role of NO and antioxidants in mitigating these effects.