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

基本信息

批准号：
10370658
负责人：
Neena Singh
金额：
$ 20.13万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10370658
关键词：
Anterior Anterior eyeball segment structure Antioxidants Aqueous Humor Blood 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 Epithelial Cells 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 Metals Modeling Monitor Mus Nature Ocular Pathology Operative Surgical Procedures Organ Culture Techniques Pathologic Patients Perfusion Physiologic Intraocular Pressure Physiological Primary Open Angle Glaucoma 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 Transferrin Up-Regulation Vitreous humor antagonist anterior chamber aqueous base clinically relevant cytokine efficacy evaluation experimental study hepcidin in vivo lens metal transporting protein 1 mouse model peptide hormone prevent stem

项目摘要

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.

抽象的铁是必不可少的生物金属，但需要严格的调节以最大程度地减少铁催化的反应性氧（ROS）。这是由肝素蛋白（主要是调节肝炎的肝炎骑马蛋白）完成的血清转铁蛋白铁（TF-IROIN）通过下调铁蛋白（FPN），一种铁出口蛋白。局部的合成纤毛上皮，角膜内皮和小梁网（TM）细胞中的肝素蛋白表明自主在前部段中铁的调节，与视网膜无关。但是，肝素已更新细胞因子也是后一个信号，从TF铁中取代信号。这引起了Cytokin-的关注介导的肝素上调，通过增加细胞内铁和ROS（已知）来创造有毒环境初级开角青光眼（POAG）和其他眼部条件中毒性的贡献者。与此相关应用是TGFβ2，一种在POAG中实现的细胞因子。我实验室的数据表明，生物活性 TGFβ2上调原代人TM细胞中的肝素和前体的人体器官培养灌注模型 Poag。值得注意的是，肝素上调全长（FL）和生物活性TGFβ2，后来可能通过ROS，形成一个自我维持的前馈环，被肝素，肝素拮抗剂和 N-乙酰基肌肽，一种抗氧化剂。基于这些观察结果，我们假设前段通过局部合成的肝素来维持铁的自主调节，并介导的细胞因子介导的上调局部肝素形成一种自我维持的前馈环，被肝素拮抗剂和FPN破坏稳定剂。该假设将以三个特定目的进行检验。在AIM 1中，铁的自主调节由前部将以野生型（WT）C57BL/6和BALB/C小鼠的稳定状态和之后探索。已知会导致定期铁积累的全身铁超负荷。血清铁在血液中的运输 - 水屏障，从视网膜到水性幽默（AH）将在不存在或存在的情况下确定 AH中的外源合成肝素。局部肝素在角膜内皮细胞中的作用在调节 AH和角膜之间的铁交换将在人角膜杯前体内模型中确定。在目标2， FL和生物活性TGFβ2的水平将在Hepcidin敲除（HEPC - / - ）和同窝式HEPC+/+中确定在稳态和过表达的FLTGFβ2之后，可以评估缺乏肝素和铁 - 是否存在介导的ROS下降的生物活性TGFβ2和IOP 。另外，肝素，TF铁和TGFβ2的水平将是在手术时收集的POAG病例和白内障对照的人类AH中确定。在AIM 3，前体内人前节器官培养灌注模型将用于评估FDA批准的FPN的效率在减少细胞内铁，ROS和生物活性TGFβ2介导的稳定剂fursultiamine中增加 IOP 。这些研究的成功完成将阐明局部肝素在调节前部铁中的作用细分及其通过铁介导的ROS对眼病理学的贡献。这些研究的临床相关性源于使用可用的肝素拮抗剂和FPN稳定剂破坏该环的潜力。