Mechanisms of Epithelial Alterations in Diabetic Cornea

糖尿病角膜上皮改变的机制

基本信息

批准号：
7289238
负责人：
Alexander V Ljubimov
金额：
$ 37.87万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7289238
关键词：
Animal Model Animals Antisense Oligonucleotides Basement membrane Blindness Bullous Keratopathy Cell Adhesion Clinical Complication Complications of Diabetes Mellitus Cornea Corneal Diseases Corneal edema Data Debridement Defect Development Diabetes Mellitus Diabetic Retinopathy Edema Endopeptidases Epithelial Epithelial-Stromal Communication Esthesia Exposure to Eye diseases Funding Gene Delivery Gene Expression Gene Transfer Genes Growth Factor Hemorrhage Hepatocyte Growth Factor Human Immunohistochemistry Impaired wound healing Keratitis Keratoconus Keratopathy Lead Mediating Membrane Membrane Proteins Microaneurysm Microarray Analysis Modeling Molecular Molecular Profiling Nerve Neuropathy Organ Culture Techniques Paper Patients Pattern Peer Review Peptide Hydrolases Permeability Phenotype Proteolysis Rate Recurrence Research Personnel Retina Retinal Retinal Detachment Retinal Neovascularization Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction System Testing Thymosin Up-Regulation Viral Vitrectomy Vitreous Hemorrhage Wound Healing base cathepsin F corneal epithelium cytokine density diabetic gene therapy human disease macular edema migration migration stimulating factor neovascularization non-diabetic programs promoter protein expression proteinase In receptor stromelysin 2 thymosin beta(4)

Animal Model Animals Antisense Oligonucleotides Basement membrane Blindness Bullous Keratopathy Cell Adhesion Clinical Complication Complications of Diabetes Mellitus Cornea Corneal Diseases Corneal edema Data Debridement Defect Development Diabetes Mellitus Diabetic Retinopathy Edema Endopeptidases Epithelial Epithelial-Stromal Communication Esthesia Exposure to Eye diseases Funding Gene Delivery Gene Expression Gene Transfer Genes Growth Factor Hemorrhage Hepatocyte Growth Factor Human Immunohistochemistry Impaired wound healing Keratitis Keratoconus Keratopathy Lead Mediating Membrane Membrane Proteins Microaneurysm Microarray Analysis Modeling Molecular Molecular Profiling Nerve Neuropathy Organ Culture Techniques Paper Patients Pattern Peer Review Peptide Hydrolases Permeability Phenotype Proteolysis Rate Recurrence Research Personnel Retina Retinal Retinal Detachment Retinal Neovascularization Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction System Testing Thymosin Up-Regulation Viral Vitrectomy Vitreous Hemorrhage Wound Healing base cathepsin F corneal epithelium cytokine density diabetic gene therapy human disease macular edema migration migration stimulating factor neovascularization non-diabetic programs promoter protein expression proteinase In receptor stromelysin 2 thymosin beta(4)

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项目摘要

DESCRIPTION: Diabetic retinopathy (DR) is a severe complication of diabetes mellitus leading to vision loss in millions of patients. The main ocular target of DR is the retina that develops intravitreal hemorrhages, macular edema, retinal detachments and neovascularization. However, 70% of diabetic patients also suffer from corneal abnormalities. Clinical corneal problems include epithelial alterations (abnormal barrier function, defects and recurrent erosions, debridement after vitrectomy, delayed wound healing, keratitis, and edema), as well as neuropathy. Altered epithelial-stromal interactions and basement membrane (BM) structure likely contribute to diabetic epitheliopathy. In the previous funding period, we have validated human corneal organ culture model because diabetic animals do not fully reproduce human diabetic eye disease. Organ-cultured corneas from DR patients retain marker abnormalities and delayed wound healing as observed in diabetic patients. Organ-cultured corneas from DR patients maintain altered BM protein expression observed in intact donor corneas. Additionally, DR corneas have decreased levels of migratory growth factors/cytokines, such as HGF/c-met system and thymosin beta4, and simultaneously increased expression of proteinases, MMP-10 and cathepsin F. Changes in growth factor expression and increased proteolysis may trigger BM and wound healing alterations leading to clinically observed diabetic corneal disease. We propose the following hypothesis: Epithelial alterations found in diabetic corneas are due to decreased levels or activity of migration stimulating factors, such as HGF and thymosin beta4, accompanied by increased levels of proteinases, such as MMP-10 and cathepsin F, resulting in enhanced epithelial BM degradation and reduced cell adhesion. These changes will lead to delayed epithelial wound healing and persistent defects. Specific correction of these alterations by antisense inhibition or viral-mediated gene transfer may alleviate diabetic corneal epitheiiopathy. To test this mechanism, we propose the following Specific Aims: Aim 1. To examine the role of growth factor alterations in diabetic corneal abnormalities using gene-based therapies in human corneal organ culture. Diabetic organ-cultured corneas will be treated with viral constructs harboring c-met or thymosin betap4 genes under a cornea-specific promoter to restore normal expression and HGF signaling through c-met receptor. Normal organ-cultured corneas will be treated with antisense oligonucleotides to cmet and thymosin beta4 to block their expression. With respect to marker patterns and wound healing rates, we expect treated DR corneas to become more similar to normal but normal corneas to become more similar to DR corneas. Aim 2. To determine the role of proteinases in diabetic corneal alterations using human corneal organ culture model. DR organ-cultured corneas will be treated with antisense oligonucleotides to MMP-10 or cathepsin F, to inhibit their expression. Normal organ-cultured corneas will be treated with viral constructs harboring cathepsin F or MMP-10 genes to increase their expression. We expect that treated DR corneas will be similar to normal but treated normal corneas will be similar to DR by marker patterns and wound healing rates. Aim 3. Demonstrate that diabetic alterations in wound healing and marker distribution result from a combined action of growth factors and proteinases. Growth factor and proteinase gene expression will be changed together towards normal in DR corneas using viral-based gene delivery and antisense approach. Expression of DR markers and wound healing rates will be determined and compared to single gene therapies. This approach will be also used in normal corneas in order to elicit delayed wound healing and DR-like changes in marker expression. These studies should facilitate the development of gene-based new therapy for diabetic corneal abnormalities.

描述：糖尿病性视网膜病（DR）是糖尿病的严重并发症，导致数百万患者的视力丧失。 DR的主要眼靶是视网膜，该视网膜发生玻璃体内出血，黄斑水肿，视网膜脱离和新生血管形成。但是，有70％的糖尿病患者也患有角膜异常。临床角膜问题包括上皮改变（异常屏障功能，缺陷和复发性侵蚀，玻璃体切除术后的清创，伤口延迟的愈合，角膜炎和水肿）以及神经病。上皮 - 层相互作用和基础膜（BM）结构可能导致糖尿病上皮病。在上一个资金期间，我们已经验证了人角膜器官培养模型，因为糖尿病动物没有完全再现人类糖尿病眼病。从糖尿病患者中观察到的，DR患者的器官培养角膜保留了标记异常和延迟伤口愈合。 DR患者的器官培养角膜维持在完整的供体角膜中观察到的BM蛋白表达改变。此外，Corneas的迁移生长因子/细胞因子的水平降低，例如HGF/C-MET系统和胸腺素β4，同时增加了蛋白酶，MMP-10和组织蛋白酶F的表达。生长因子表达和增加的蛋白水解的变化可能会触发BM和伤口造成BM和伤害愈合的疾病，从而导致临床上观察到的糖尿病性疾病。我们提出以下假设：糖尿病角膜中发现的上皮改变是由于迁移刺激因子的水平或活性降低，例如HGF和胸腺素β4，伴随着蛋白酶水平的升高，例如MMP-10和天息肉F，导致上皮BM脱发和降低的细胞降低。这些变化将导致上皮伤口愈合和持续缺陷。通过反义抑制或病毒介导的基因转移对这些改变的特定校正可能会减轻糖尿病角膜上皮病。为了测试这种机制，我们提出了以下特定目的：目的1。使用基于基因的人角膜器官培养中基因疗法来检查生长因子改变在糖尿病角膜异常中的作用。糖尿病器官培养的角膜将通过角膜特异性启动子下的含有C-MET或胸腺素BETAP4基因的病毒构建体处理，以通过C-MET受体恢复正常表达和HGF信号传导。正常的器官培养角膜将用反义寡核苷酸和胸腺素β4进行处理以阻断其表达。关于标记模式和伤口愈合率，我们预计经过治疗的Corneas博士会变得与正常但正常角膜更相似，与Corneas博士更相似。目的2。使用人角膜器官培养模型来确定蛋白酶在糖尿病角膜改变中的作用。器官培养的角膜将用反义寡核苷酸与MMP-10或组织蛋白酶F处理，以抑制其表达。正常的器官培养的角膜将用携带组织蛋白酶F或MMP-10基因的病毒构建体进行处理，以增加其表达。我们预计经过处理的DR角膜将与正常的角膜相似，但是经过治疗的正常角膜将与Marker模式和伤口愈合率相似。目标3。证明伤口愈合和标记分布的糖尿病改变是由生长因子和蛋白酶的综合作用引起的。生长因子和蛋白酶基因表达将使用基于病毒的基因递送和反义方法在DR角膜中正常变化。将确定DR标记和伤口愈合率的表达并将其与单个基因疗法进行比较。这种方法还将用于正常角膜，以引起延迟的伤口愈合和标记表达的DR样变化。这些研究应促进基于基因的糖尿病角膜异常疗法的发展。