Mechanisms of Epithelial Alterations in Diabetic Cornea

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的主要眼部靶点是视网膜，其发展为玻璃体内水肿、黄斑水肿、视网膜脱离和新生血管。然而，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（屏障功能异常、缺陷和复发性糜烂、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。改变的上皮-基质相互作用和基底膜（BM）结构可能有助于糖尿病上皮病变。 在上一个资助期，我们已经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标记物异常和延迟的伤口愈合，如在糖尿病患者中观察到的。来自DR患者的器官培养的角膜保持在完整供体角膜中观察到的改变的BM蛋白表达。此外，DR角膜的迁移性生长因子/细胞因子水平降低，如HGF/c-met系统和胸腺素β 4，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的增加可能触发BM和伤口愈合的改变，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（如HGF和胸腺素β 4）的水平或活性降低，伴随蛋白酶（如MMP-10和组织蛋白酶F）水平升高，导致上皮BM降解增强和细胞粘附减少。这些变化将导致延迟的上皮伤口愈合和持续的缺陷。 通过反义抑制或病毒介导的基因转移特异性纠正这些改变可能会减轻糖尿病性角膜上皮病。为了测试这种机制，我们提出了以下具体目标：目标1。在人角膜器官培养中使用基于基因的治疗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用在角膜特异性启动子下含有c-met或胸腺素β 4基因的病毒构建体处理，以恢复正常表达和通过c-met受体的HGF信号传导。将用cmet和胸腺素β 4的反义寡核苷酸处理正常器官培养的角膜以阻断其表达。关于标记模式和伤口愈合率，我们预期经治疗的DR角膜变得与正常角膜更相似，但正常角膜变得与DR角膜更相似。 目标二。利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。将用携带组织蛋白酶F或MMP-10基因的病毒构建体处理正常器官培养的角膜以增加其表达。我们预计经治疗的DR角膜将与正常角膜相似，但经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：证明糖尿病患者伤口愈合和标志物分布的改变是生长因子和蛋白酶共同作用的结果。使用基于病毒的基因递送和反义方法，生长因子和蛋白酶基因表达将一起在DR角膜中向正常改变。将确定DR标志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，以引起延迟的伤口愈合和标记物表达的DR样变化。 这些研究将促进糖尿病角膜病变基因治疗的发展。

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.