Mechanisms of Epithelial Alterations in Diabetic Cornea

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的主要眼部目标是视网膜，视网膜会发生玻璃体内出血、黄斑水肿、视网膜脱离和新生血管。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。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变。在之前的资助期内，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性眼病。在糖尿病患者中观察到，DR患者的器官培养角膜保留了标志物异常和伤口愈合延迟。来自DR患者的器官培养角膜在完整的供体角膜中观察到BM蛋白表达的改变。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系统和胸腺酶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引发BM和伤口愈合的改变，从而导致临床观察到的糖尿病性角膜疾病。我们提出以下假设：在糖尿病角膜中发现的上皮改变是由于迁移刺激因子（如HGF和胸腺蛋白酶β 4）的水平或活性降低，伴随着蛋白酶（如MMP-10和组织蛋白酶F）水平的升高，导致上皮BM降解增强，细胞粘附减少。这些变化将导致上皮性伤口愈合延迟和持续缺损。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纠正可能会减轻糖尿病性角膜上皮病。为了验证这一机制，我们提出以下具体目标：在人角膜器官培养中使用基于基因的治疗方法研究生长因子改变在糖尿病角膜异常中的作用。在角膜特异性启动子下，用含有c-met或胸腺酶β ap4基因的病毒构建物治疗糖尿病器官培养角膜，恢复正常表达和通过c-met受体的HGF信号传导。正常器官培养角膜将用反义寡核苷酸和胸腺酶β 4来阻断它们的表达。在标记模式和伤口愈合率方面，我们预计治疗后的DR角膜会变得更接近正常角膜，而正常角膜会变得更接近DR角膜。目标2。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器官培养角膜用MMP-10或组织蛋白酶F的反义寡核苷酸处理，抑制其表达。正常器官培养的角膜将用含有组织蛋白酶F或MMP-10基因的病毒构建体处理，以增加其表达。我们预计，治疗后的DR角膜将与正常角膜相似，但治疗后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记物分布的改变是生长因子和蛋白酶共同作用的结果。利用基于病毒的基因传递和反义方法，将生长因子和蛋白酶基因表达共同向正常方向改变。将确定DR标记物的表达和伤口愈合率，并与单基因治疗进行比较。这种方法也将用于正常角膜，以引起延迟伤口愈合和标记物表达的dr样变化。这些研究将促进基于基因的糖尿病角膜异常新疗法的发展。

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.