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：
7663781
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 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7663781
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 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 Principal Investigator Proteolysis Recurrence 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 diabetic patient gene therapy human disease macular edema meetings migration migration stimulating factor neovascularization non-diabetic programs promoter protein expression proteinase In receptor stromelysin 2 thymosin beta(4)

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：糖尿病视网膜病变（DR）是糖尿病的严重并发症，导致数百万患者视力丧失。 DR 的主要眼部靶标是视网膜，导致玻璃体内出血、黄斑水肿、视网膜脱离和新生血管形成。然而，70%的糖尿病患者同时患有角膜异常。临床角膜问题包括上皮改变（屏障功能异常、缺损和反复糜烂、玻璃体切除术后清创、伤口愈合延迟、角膜炎和水肿）以及神经病变。上皮间质相互作用和基底膜（BM）结构的改变可能导致糖尿病上皮病。在之前的资助期间，我们验证了人类角膜器官培养模型，因为糖尿病动物不能完全复制人类糖尿病眼病。 DR 患者的器官培养角膜保留了标记异常，并且伤口愈合延迟，正如在糖尿病患者中观察到的那样。 DR 患者的器官培养角膜维持了在完整供体角膜中观察到的改变的 BM 蛋白表达。此外，DR角膜的迁移生长因子/细胞因子（例如HGF/c-met系统和胸腺素β4）水平降低，同时蛋白酶、MMP-10和组织蛋白酶F的表达增加。生长因子表达的变化和蛋白水解作用的增加可能会引发BM 和伤口愈合改变导致临床观察到的糖尿病性角膜疾病。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（例如 HGF 和胸腺素 β4）水平或活性降低，同时伴随着蛋白酶（例如 MMP-10 和组织蛋白酶 F）水平增加，导致迁移刺激因子增强上皮BM降解和细胞粘附减少。这些变化将导致上皮伤口愈合延迟和持续缺陷。通过反义抑制或病毒介导的基因转移对这些改变进行特异性校正可以减轻糖尿病性角膜上皮病变。为了测试这一机制，我们提出以下具体目标：目标 1. 在人类角膜器官培养中使用基于基因的疗法来检查生长因子改变在糖尿病角膜异常中的作用。将用在角膜特异性启动子下含有 c-met 或胸腺素 betap4 基因的病毒构建体处理糖尿病器官培养的角膜，以通过 c-met 受体恢复正常表达和 HGF 信号传导。正常器官培养的角膜将用 cmet 和胸腺素 beta4 的反义寡核苷酸处理，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 DR 角膜会变得与正常角膜更加相似，但正常角膜会变得与 DR 角膜更加相似。目标 2. 使用人角膜器官培养模型确定蛋白酶在糖尿病角膜改变中的作用。 DR 器官培养的角膜将用 MMP-10 或组织蛋白酶 F 的反义寡核苷酸处理，以抑制其表达。正常的器官培养角膜将用含有组织蛋白酶 F 或 MMP-10 基因的病毒构建体进行处理，以增加其表达。我们预计经过治疗的 DR 角膜将与正常角膜相似，但经过治疗的正常角膜在标记模式和伤口愈合率方面将与 DR 相似。目标 3. 证明糖尿病对伤口愈合和标记物分布的改变是生长因子和蛋白酶的联合作用的结果。使用基于病毒的基因传递和反义方法，DR 角膜中的生长因子和蛋白酶基因表达将一起改变为正常。将确定 DR 标记物的表达和伤口愈合率，并与单基因疗法进行比较。这种方法也将用于正常角膜，以引起伤口愈合延迟和标志物表达的类似 DR 的变化。这些研究应有助于开发基于基因的糖尿病角膜异常新疗法。