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（雾兹）每年130万美国人失明占世界失明的7％。目前的药物通常无法治愈严重和确定的角膜雾。目前，角膜移植手术是护理标准。普通的透明角膜不包含肌纤维细胞。侮辱后，通过转化生长因子B激活静止透明的角膜细胞（TGFB）成为角膜基质成纤维细胞（CSF），然后将其转分化成角膜肌纤维细胞（CMFS）进行角膜修复。在早期和持久性期间CMF的过度和长时间形成在伤口愈合的后期，基质中的CMF是雾化的主要原因。最近，我们发现了2种调节这一过程的新机制：（a）雾霾产生涉及中间电导Ca2+激活的K+通道，KCA3.1，而（B）在体内消除（B）雾度消除可以通过选择性诱导通过双重疗法在CMF中凋亡。这项研究提供了第一个证据，表明可以通过指示雾化 CMFS在体内的命运。 CMF不是终端分化的细胞，可以在体外恢复为CSF。肌成纤维细胞的形成，逆转和脱不同是组织特异性事件，没有角膜特异性数据目前存在。这个前提导致了一个中心假设，即新型的表观遗传方法用于指导命运 CMF的体内将消除体内既定且严重的雾度，并提供非手术的视力手段恢复。该项目旨在优化非手术方法来治疗雾霾，以追求3个特定目标。 AIM-1测试 TRAM-34（KCA3.1特异性抑制剂）在伤口愈合的早期阶段限制了CMF形成的假设通过上调限制性元素1沉默转录来抑制KCNN4基因转录因子（静止）还原AP1（激活蛋白-1）。 AIM-2检验了表观遗传重编程的假设丁酸钠（NAB）伤口愈合的后期愈合将有效地脱离CMFS前体CSFS 通过增强DNA甲基转移（DNMT），甲基CpG结合蛋白2的体外体外和角膜细胞（MECP2）和CpG岛上CpG岛上α-光滑肌肉肌动蛋白基因上的CpG岛的DNA甲基化。 AIM-3测试一个假设，即Tram-34+NAB双重疗法将完全废除兔子中的体内严重/已建立的雾度（a）限制TGFB驱动的过量和延长的CMF形成的显着副作用通过减少TRAM-34的KCA3.1的伤口愈合，（b）促进CMF在后期的前体细胞逆转到前体细胞 NAB通过表观遗传重编程通过伤口愈合。我们的试点研究强烈支持假设。建议的研究将采用既定的体外人和体内（兔子和kca3.1 - / - 小鼠）角膜来完成研究纤维化模型，临床眼检查，分子和细胞测定法，并遵循我们发布的方法。项目的成功结论将填补关键知识空白，并大大提高角膜领域。