Novel approaches for corneal haze/fibrosis elimination

消除角膜混浊/纤维化的新方法

基本信息

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
项目类别：
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

项目摘要

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.

抽象的受伤、感染或创伤后的角膜纤维化（雾霾）每年导致 130 万美国人失明，世界上 7% 的失明患者都是由这种疾病引起的，目前的药物往往无法治愈严重且已形成的角膜混浊。目前，角膜移植手术是标准的护理方法，正常的透明角膜不含任何成分。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，在早期和持续阶段过度且长时间地形成 CMF。最近，我们发现伤口愈合后期基质中的 CMF 是体内雾霾的主要原因。发现了调节这一过程的 2 个新机制：(a) 雾霾的产生涉及中间- 电导 Ca2+ 激活的 K+ 通道，KCa3.1，而 (b) 体内雾霾消除可以通过选择性诱导通过双重疗法促进 CMF 细胞凋亡这项研究提供了第一个证据，表明雾霾可以通过直接清除。 CMF在体内的命运不是终末分化细胞，在体外可以逆转为CSF。肌成纤维细胞形成、逆转和去分化是组织特异性事件，没有角膜特异性数据这个前提导致了一个中心假设，即新的表观遗传学方法可以用来指导命运。体内 CMF 将消除体内已形成的严重雾霾，并提供非手术的视觉手段该项目旨在优化非手术方法来治疗雾霾，实现 3 个具体目标。假设 TRAM-34（一种 KCa3.1 特异性抑制剂）限制伤口愈合早期阶段的 CMF 形成通过上调限制性元件 1 沉默转录抑制 KCNN4 基因转录因子（REST）减少AP1（激活蛋白-1）测试了表观遗传重编程的假设。丁酸钠（NaB）在伤口愈合的后期将有效地将 CMF 去分化为前体 CSF 通过增强 DNA 甲基转移酶 (DNMT)、甲基 CpG 结合蛋白 2 在体外和体内的角膜细胞 (MeCP2) 和 α-平滑肌肌动蛋白 Aim-3 基因启动子上 CpG 岛的 DNA 甲基化。假设 TRAM-34+NaB 双重疗法将完全消除兔子体内严重/已形成的雾霾，而无需通过 (a) 在早期阶段限制 TGFb 驱动的过度和长时间的 CMF 形成来产生显着的副作用通过 TRAM-34 减少 KCa3.1 来促进伤口愈合，以及 (b) 在晚期促进 CMF 逆转为前体细胞 NaB 通过表观遗传重编程促进伤口愈合。我们的初步研究有力地支持了所提出的假设。研究将使用已建立的体外人类和体内（兔子和 KCa3.1-/- 小鼠）角膜来完成纤维化模型、临床眼科检查、分子和细胞测定以及遵循我们发布的方法。项目的成功完成将填补关键知识空白并显着推进角膜领域的发展。