Local hepcidin in the anterior segment: Physiological and pathological implications

摘要 铁是一种必需的生物金属，但需要严格的监管，以最大限度地减少铁催化的反应的产生。 氧物种(ROS)。这是由海普西丁完成的，海普西丁是一种主要的肝多肽激素，调节 通过下调铁输出蛋白铁蛋白(FPN)来调节血清转铁蛋白铁(Tf-Fe)。局部结构的合成 睫状体上皮、角膜内皮和小梁网(TM)细胞中的海普西丁提示自主性 调节前段的铁，独立于视网膜。然而，海普西丁是上调的 细胞因子也一样，后者的信号取代了来自Tf-Iron的信号。这引发了对细胞因子的担忧-- 调节海普西丁的表达，通过增加细胞内的铁和ROS来创造一个有毒的环境，已知 原发性开角型青光眼(POAG)和其他眼部疾病的毒性因素。与此相关 应用转化生长因子β-2，一种与POAG有关的细胞因子。来自我的实验室的数据显示，生物活性 转化生长因子β-2上调人TM原代细胞和体外人器官培养灌流模型 普阿格。值得注意的是，海普西丁上调全长(FL)和生物活性转化生长因子β2，后者可能 通过ROS，形成一个自我维持的前馈环路，该环路被肝素，一种海普西丁拮抗剂破坏，以及 N-乙酰肌肽，一种抗氧化剂。基于这些观察结果，我们假设眼前节 通过局部合成的海普西丁维持铁的自主调节，以及细胞因子介导的 局部海普西丁形成一个自我维持的前馈环路，可被海普西丁拮抗剂和FPN干扰 稳定剂。这一假设将在三个具体目标上得到检验。在目标1中，自主调节铁 将在野生型(Wt)C57BL/6和Balb/c小鼠的稳态和之后的 全身性铁超载，已知会导致视网膜铁积聚。血清铁在血液中的运输- 房水屏障，以及从视网膜到房水(AH)将在没有或存在的情况下进行测定 外源合成的海普西丁。局部海普西丁在角膜内皮细胞调节中的作用 AH和角膜之间的铁交换将在人体角膜杯体外模型中确定。在……里面 目标2， FL和生物活性转化生长因子β-2的水平将在海普西丁基因敲除(HEPC-/-)和产仔鼠HEPC+/+中测定 对照组在稳态，过表达FL转化生长因子β-2后，评估是否缺乏海普西丁和铁- 介导的ROS降低生物活性转化生长因子β-2和眼压 。此外，海普西丁、转铁蛋白和转化生长因子β2的水平将是 在手术收集的POAG病例和白内障对照的人类AH中测定。在目标3中，体外人类 眼前节器官培养灌流模型将用于评估FDA批准的FPN的疗效 稳定剂呋喃硫胺降低细胞内铁、ROS和生物活性转化生长因子β-2的作用 增加 眼压 。这些研究的成功完成将阐明局部海普西丁在调节前角铁质中的作用。 片段，及其通过铁介导的ROS在眼部病理中的贡献。这些研究的临床相关性 源于可用海普西丁拮抗剂和FPN稳定剂破坏这一环路的可能性。

ABSTRACT Iron is an essential bio-metal, but requires tight regulation to minimize the generation of iron-catalyzed reactive oxygen species (ROS). This is accomplished by hepcidin, a mainly hepatic peptide hormone that regulates serum transferrin iron (Tf-iron) by downregulating ferroportin (Fpn), an iron export protein. The synthesis of local hepcidin in the ciliary epithelial, corneal endothelial, and trabecular meshwork (TM) cells suggests autonomous regulation of iron in the anterior segment, independent of the retina. However, hepcidin is upregulated by cytokines as well, the latter signal superseding the signal from Tf-iron. This raises the concern of cytokine- mediated upregulation of hepcidin, creating a toxic environment by increasing intracellular iron and ROS, known contributors of toxicity in primary open angle glaucoma (POAG) and other ocular conditions. Pertinent to this application is TGFβ2, a cytokine implicated in POAG. Data from my laboratory show that biologically active TGFβ2 upregulates hepcidin in primary human TM cells and ex-vivo human organ culture perfusion model of POAG. Remarkably, hepcidin upregulates full-length (FL) and biologically active TGFβ2, the latter probably through ROS, forming a self-sustained feed-forward loop that is disrupted by heparin, a hepcidin antagonist, and N-acetyl carnosine, an antioxidant. Based on these observations, we hypothesize that the anterior segment maintains autonomous regulation of iron by locally synthesized hepcidin, and cytokine-mediated upregulation of local hepcidin forms a self-sustained feed-forward loop that is disrupted by hepcidin antagonists and Fpn stabilizing agents. This hypothesis will be tested in three specific aims. In Aim 1,autonomous regulation of iron by the anterior segment will be explored in wild-type (wt) C57BL/6 and Balb/c mice at steady state and after systemic iron overload that is known to cause retinal iron accumulation. Transport of serum iron across the blood- aqueous barrier, and from the retina to the aqueous humor (AH) will be determinedin the absence or presence of exogenous synthetic hepcidin in the AH. The role of local hepcidin in corneal endothelial cells in regulating iron exchange between the AH and the cornea will be determined in the human corneal cup ex-vivo model. In Aim 2, levels of FL and bioactive TGFβ2 will be determined in hepcidin knock-out (hepc-/-) and littermate hepc+/+ controls at steady state, and after over-expressing FL TGFβ2 to evaluate whether absence of hepcidin and iron- mediated ROS decreases bioactive TGFβ2 and IOP . In addition, levels of hepcidin, Tf-iron, and TGFβ2 will be determined in the human AH of POAG cases and cataract controls collected at surgery. In Aim 3, ex-vivo human anterior segment organ culture perfusion model will be used to evaluate the efficacy of FDA approved Fpn stabilizing agent fursultiamine in reducing intracellular iron, ROS, and bioactive TGFβ2-mediated increase in IOP . Successful completion of these studies will clarify the role of local hepcidin in regulating iron in the anterior segment, and its contribution to ocular pathology through iron-mediated ROS. Clinical relevance of these studies stems from the potential of disrupting this loop with available hepcidin antagonists and Fpn stabilizing agents.