Novel approaches for corneal haze/fibrosis elimination

消除角膜混浊/纤维化的新方法

• 批准号: 10005368
• 负责人: Rajiv Ravindra Mohan
• 金额: $ 38.48万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005368
• 关键词: Affect; Alkalies; American; Apoptosis; BMP7 gene; Binding Proteins; Blindness; Calcium-Activated Potassium Channel; Cell Differentiation process; Cellular Assay; Cicatrix; Clinical; Complement Factor B; Confocal Microscopy; Cornea; CpG Islands; DNA; DNA Methylation; DNA Modification Methylases; Data; Development; Dose; Elements; Epigenetic Process; Event; Eye; Fibroblasts; Fibrosis; Gene Silencing; Genes; Genetic Transcription; Goals; Human; Imaging Device; In Vitro; Infection; Injury; Keratoplasty; Knowledge; Lead; Mediating; Membrane; Methods; Methyl-CpG-Binding Protein 2; Microscopy; Modeling; Molecular; Mus; Myofibroblast; Operative Surgical Procedures; Ophthalmic examination and evaluation; Oryctolagus cuniculus; Parents; Pharmaceutical Preparations; Pharmacology; Phase II Clinical Trials; Pilot Projects; Process; Production; Protein Methylation; Protocols documentation; Public Health; Publishing; Reporting; Role; Smooth Muscle Actin Staining Method; Sodium Butyrate; Stress Fibers; Testing; Tissue Differentiation; Toxic effect; Transcription Factor AP-1; Transferase; Transforming Growth Factors; Transplantation Surgery; Trauma; Up-Regulation; Vision; Western Blotting; clinical examination; confocal imaging; corneal epithelial wound healing; corneal regeneration; corneal repair; corneal scar; healing; human model; in vivo; inhibitor/antagonist; irritation; novel; novel strategies; novel therapeutics; precursor cell; prevent; promoter; pyrosequencing; restoration; side effect; standard of care; transcription factor; transdifferentiation; wound closure; wound healing

Abstract Corneal fibrosis (haze) after injury, infection, or trauma causes blindness in 1.3 million Americans each year and accounts for 7% of the world’s blindness. Current drugs often fail to cure severe and established corneal haze. At present, cornea transplant surgery is the standard of care. A normal clear cornea do not contain myofibroblasts. After insult, quiescent transparent keratocytes are activated by transforming growth factor b (TGFb) to become corneal stromal fibroblasts (CSFs), which then transdifferentiate into corneal myofibroblasts (CMFs) to do corneal repair. Excessive and prolonged formation of CMFs during early-stages and persistence of CMFs in stroma in late-stages of wound healing are the primary causes of haze in vivo. Recently, we uncovered 2 novel mechanisms regulating this process: (a) haze production involves the intermediate- conductance Ca2+-activated K+ channel, KCa3.1 whereas (b) haze elimination in vivo can be induced via selective apoptosis in CMFs by a dual therapy. This study provided the first evidence that haze can be cleared by directing the fate of CMFs in vivo. CMFs are not terminally differentiated cells and can be reverted to CSFs in vitro. Myofibroblast formation, reversal, and de-differentiation are tissue-specific events, and no cornea-specific data exists currently. This premise led to a central hypothesis that novel epigenetic approaches used to direct the fate of CMFs in vivo will eliminate established and severe haze in vivo, and provide a non-surgical means of vision restoration. This project aims optimizing non-surgical method to treat haze pursuing 3 specific aims. Aim-1 tests a hypothesis that TRAM-34 (a KCa3.1 specific inhibitor) limits CMF formation in early-stages of wound healing in vivo by inhibiting KCNN4 gene transcription via upregulation of Restrictive element-1 silencing transcription factor (REST) reducing AP1 (Activator protein-1). Aim-2 tests a hypothesis that epigenetic reprogramming in late-stages of wound healing by Sodium butyrate (NaB) will efficiently de-differentiate CMFs to precursor CSFs in vitro and keratocytes in vivo by enhancing DNA methyl transferases (DNMTs), methyl CpG binding protein 2 (MeCP2) and DNA methylation of CpG islands on the promoter on alpha-smooth muscle actin gene. Aim-3 tests a hypothesis that TRAM-34+NaB dual therapy will fully abolish severe/established haze in vivo in rabbits without significant side effects by (a) limiting TGFb-driven excessive and prolonged CMFs formation in early-stage wound healing by reducing KCa3.1 by TRAM-34, and (b) promoting CMFs reversal to precursor cells in late-stage wound healing by NaB via epigenetic reprogramming. Our pilot studies strongly support hypotheses. Proposed studies will be accomplished employing established in vitro human and in vivo (rabbit and KCa3.1-/- mice) corneal fibrosis models, clinical eye exams, molecular, and cellular assays and following our published method. The successful conclusion of project will fill key knowledge gaps and significantly advance the corneal field.

摘要 损伤、感染或创伤后的角膜纤维化(Haze)每年导致130万美国人失明， 占全球失明人数的7%。目前的药物往往不能治愈严重的和已建立的角膜混浊。 目前，角膜移植手术是护理的标准。正常的透明角膜不含 肌成纤维细胞。损伤后，静止的透明角膜细胞被转化生长因子b激活。 (TGFb)成为角膜基质成纤维细胞(CSF)，然后转分化为角膜肌成纤维细胞 (CMF)进行角膜修复。CMF在早期和持续性中的过度和长期形成 创面愈合后期基质中CMF的大量存在是造成体内雾霾的主要原因。最近，我们 发现了两种调节这一过程的新机制：(A)雾霾的产生涉及中间体- 电导钙激活的K+通道，KCa3.1，而(B)体内雾霾的消除可以通过选择性地诱导 通过双重疗法使巨噬细胞发生细胞凋亡。这项研究提供了第一个证据，表明雾霾可以通过指导 CMF在体内的命运。CMF不是终末分化的细胞，在体外可以转化为CSF。 肌成纤维细胞的形成、逆转和去分化是组织特有的事件，没有角膜特有的数据。 当前存在。这一前提导致了一个中心假设，即用新的表观遗传学方法来指导命运 在体内的CMF将消除体内已建立的和严重的雾霾，并提供一种非手术的视觉手段 修复。本项目旨在优化非手术治疗雾霾的方法，追求3个具体目标。AIM-1测试 TRAM-34(一种KCa3.1特异性抑制剂)在创伤愈合早期限制CMF形成的假说 通过上调限制性元件-1沉默转录抑制KCNN4基因转录 因子(REST)降低AP1(激活蛋白-1)。AIM-2测试了一种假设，即表观遗传重新编程在 丁酸钠(NAB)在创面愈合的后期将有效地将CMF与前体CSF区分开来 DNA甲基转移酶(DNMT)、甲基CpG结合蛋白2在体外和体内对角膜基质细胞的增强作用 (MeCP2)和α-平滑肌肌动蛋白基因启动子上CpG岛的DNA甲基化。AIM-3测试 TRAM-34+NAB双重治疗将在体内完全消除严重/已建立的雾霾 通过(A)在早期阶段限制TGFb驱动的过量和长期的CMF形成而产生的显著副作用 TRAM-34通过降低KCa3.1促进创面愈合，以及(B)促进CMF在晚期逆转为前体细胞 NAB通过表观遗传重新编程实现伤口愈合。我们的初步研究有力地支持了这些假设。建议 研究将使用建立的体外人和活体(兔和KCa3.1-/-小鼠)角膜 纤维化模型，临床眼科检查，分子和细胞分析，并遵循我们发表的方法。这个 项目的成功完成将填补关键知识空白，并显著推进角膜领域的发展。