Local hepcidin in the anterior segment: Physiological and pathological implications

眼前节局部铁调素：生理和病理学意义

• 批准号: 10546487
• 负责人: Neena Singh
• 金额: $ 24.15万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10546487
• 关键词: Anterior; Anterior eyeball segment structure; Antioxidants; Aqueous Humor; Blood-Aqueous Barrier; Blood-Retinal Barrier; Carnosine; Cataract; Cattle; Cells; Chemistry; Ciliary epithelium; Clinical Trials; Contralateral; Cornea; Corneal Endothelium; Data; Disease; Down-Regulation; Endothelial Cells; Environment; Epithelial Cells; Epithelium; Eye; FDA approved; Ferritin; Generations; Glaucoma; Harvest; Heparin; Hepatic; Homeostasis; Human; In Transferrin; Inbred BALB C Mice; Interleukin-6; Iron; Iron Overload; Knock-out; Laboratories; Length; Lipopolysaccharides; Measures; Mediating; Metabolic; Modeling; Monitor; Mus; Nature; Ocular Pathology; Operative Surgical Procedures; Organ Culture Techniques; Pathologic; Patients; Perfusion; Physiologic Intraocular Pressure; Physiological; Primary Open Angle Glaucoma; Probability; Protein Export Pathway; ROS1 gene; Reaction; Reactive Oxygen Species; Regulation; Reporting; Retina; Role; Sampling; Serum; Signal Transduction; Stabilizing Agents; Structure; Testing; Therapeutic; Time; Toxic effect; Trabecular meshwork structure; Trace Elements; Transfection; Transferrin; Transforming Growth Factor beta; Up-Regulation; Vitreous humor; antagonist; anterior chamber; clinically relevant; cytokine; efficacy evaluation; experimental study; hepcidin; in vivo; lens; metal transporting protein 1; mouse model; overexpression; peptide hormone; prevent; stem

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.

摘要 铁是一种重要的生物金属，但需要严格的调控，以尽量减少铁催化反应的产生。 氧物种（ROS）。这是通过铁调素完成的，铁调素是一种主要的肝肽激素， 血清转铁蛋白铁（Tf-铁）通过下调铁转运蛋白（Fpn），铁输出蛋白。地方综合 睫状体上皮、角膜内皮和小梁网（TM）细胞中的铁调素提示自主的 调节眼前节中的铁，独立于视网膜。然而，铁调素是上调 细胞因子以及，后者的信号取代信号从Tf-铁。这引起了对细胞因子的关注- 介导的铁调素上调，通过增加细胞内铁和ROS产生毒性环境， 在原发性开角型青光眼（POAG）和其他眼部疾病中，与此相关 应用是TGFβ2，一种与POAG有关的细胞因子。我实验室的数据显示 TGFβ2在原代人TM细胞和离体人器官培养灌注模型中上调铁调素 POAG。值得注意的是，hepcidin上调全长（FL）和生物活性TGFβ2，后者可能 通过ROS，形成一个自我维持的前馈回路，该回路被肝素（一种铁调素拮抗剂）破坏， N-乙酰肌肽，抗氧化剂。基于这些观察，我们假设眼前节 通过局部合成的铁调素维持铁的自主调节，并且马槟榔碱介导的 局部铁调素形成自我维持的前馈环，其被铁调素拮抗剂和Fpn破坏 稳定剂。这一假设将在三个具体目标中得到检验。在目标1中，铁的自主调节 将在野生型（wt）C57 BL/6和Balb/c小鼠中在稳态和稳态后 已知引起视网膜铁积聚的全身性铁过载。血清铁在血液中的运输- 将在不存在或存在下测定从视网膜到房水（AH）的房水屏障， 外源性合成铁调素角膜内皮细胞局部hepcidin在调节角膜内皮细胞凋亡中的作用 AH和角膜之间的铁交换将在人角膜杯离体模型中测定。在 目标二， 将在铁调素敲除（hepc-/-）和同窝hepc+/+中测定FL和生物活性TGFβ2的水平 稳定状态下的对照，以及过表达FL TGFβ2后，以评估铁调素和铁- ROS介导的TGFβ2降低眼压 .此外，铁调素、Tf-铁和TGFβ2的水平将被检测。 在手术时收集的POAG病例和白内障对照的人类AH中确定。在目标3中，离体人 眼前节器官培养灌注模型将用于评价FDA批准的Fpn的疗效 稳定剂呋喃硫胺在减少细胞内铁、ROS和生物活性TGFβ2介导的 增加 IOP .这些研究的成功完成将阐明局部铁调素在调节前额叶铁中的作用。 段，其通过铁介导的ROS对眼部病理的贡献。这些研究的临床相关性 来源于可用的铁调素拮抗剂和Fpn稳定剂破坏该环的潜力。