Mechanisms of Epithelial Alterations in Diabetic Cornea

描述(申请人提供)：糖尿病视网膜病变为首位 自1974年以来，美国的失明原因。这一点体现在 糖尿病眼的微血管进行性变化，导致 玻璃体内出血、视网膜水肿、新生血管和脱离。 除了视网膜，角膜、晶状体和虹膜也会受到糖尿病的影响。 许多糖尿病患者患有糖尿病角膜病变，包括复发性 糜烂、上皮脆性、创面愈合异常和增多 易受伤害。改变的上皮-间质相互作用和 上皮基底膜(BM)缺陷可能与糖尿病有关 角膜病。尽管糖尿病角膜疾病具有重要的临床意义，但 这种并发症的分子机制尚不清楚。 在我们的初步研究中，许多BM成分和 对正常人和糖尿病人的角膜进行了蛋白水解酶的分析。我们展示了 1.糖尿病视网膜病变(DR)角膜上皮细胞严重减少 BM对层粘连蛋白-1、层粘连蛋白-10、Nidogen-1/entactin的免疫染色，以及对 上皮整合素α3β1；2.BM蛋白基因表达和 糖尿病患者和DR患者角膜上皮细胞中整合素α3β1无明显变化； 基质金属蛋白酶-10的基因和蛋白表达增加 糖尿病和糖尿病视网膜病变的角膜上皮和基质，以及基质金属蛋白酶-3的表达增加 在糖尿病和DR角膜基质中。数据表明，主要成分 糖尿病尤其是糖尿病视网膜病变患者角膜上皮BM改变 特定蛋白水解酶的活性，例如，在细胞中表达的基质金属蛋白酶-10 上皮组织。 我们的假设是糖尿病和糖尿病视网膜病变患者的角膜上皮BM 通过升高的蛋白水解酶降解，尤其是通过基质金属蛋白酶-10。蛋白酶的表达 糖尿病患者激活的特定生长因子可能会刺激活性 微环境。这些改变可能构成了糖尿病的分子机制。 糖尿病患者的角膜上皮异常。 具体目的1.研究基质金属蛋白酶-10对角膜完整性的影响 上皮BM和整合素α_3β_1与器官培养的伤口愈合 人类的眼角膜。 特定目的2.用基因芯片分析鉴定生长因子和细胞因子 在糖尿病患者和DR患者的角膜中异常表达，并检测其对 基质金属蛋白酶-10与正常器官培养的角膜伤口愈合 具体目的3.通过基因芯片分析评估 糖尿病人、糖尿病视网膜病变和正常人角膜中的各种蛋白。确定和 分析糖尿病角膜中表达升高的其他蛋白。 特定目的4.试图阻断人骨髓和整合素的降解 糖尿病和DR器官培养角膜。特异性中和抗体 生长因子和蛋白酶(主要是基质金属蛋白酶-10)，以及各种基质金属蛋白酶抑制剂 包括临床批准的四环素类药物将在器官培养中进行测试 眼角膜。这些研究可能导致新疗法的开发， 会阻止糖尿病角膜病变的进展。

DESCRIPTION (provided by applicant): Diabetic retinopathy has been the leading cause of blindness in the United States since 1974. It is manifest by progressive changes in the microvasculature of the diabetic eye, leading to intravitreal hemorrhages, retinal edema, neovascularization, and detachments. Along with the retina, cornea, lens and iris are also affected by diabetes. Many diabetics suffer from diabetic keratopathy that includes recurrent erosions, epithelial fragility, abnormal wound healing and increased susceptibility to injury. Altered epithelial-stromal interactions and epithelial basement membrane (BM) defects likely contribute to diabetic keratopathy. Despite clinical importance of diabetic corneal disease, the molecular mechanisms of this complication are not understood. In our preliminary studies, the expression of many BM components and proteinases has been analyzed in normal and diabetic human corneas. We show that: 1. Diabetic retinopathy (DR) corneas have severely decreased epithelial BM immunostaining for laminin-1, laminin-10, nidogen-1/entactin, and for epithelial integrin alpha3 beta1; 2. Gene expression of BM proteins and integrin alpha3 beta1 is not changed in diabetic and DR corneal epithelium; 3. Gene and protein expression of matrix metalloproteinase (MMP)-10 increases in diabetic and DR corneal epithelium and stroma, and MMP-3 expression increases in diabetic and DR corneal stroma. The data suggest that major components of corneal epithelial BM are altered in diabetes and especially DR due to elevated activity of specific proteinases, e.g., of MMP-1O that is expressed in the epithelium. Our hypothesis is that corneal epithelial BM in diabetes and DR undergoes degradation by elevated proteinases, notably by MMP-10. Proteinase expression and activity may be stimulated by specific growth factors activated by diabetic microenvironment. These alterations may constitute the molecular mechanism of corneal epithelial abnormalities in diabetes. Specific Aim 1.To characterize the effect of MMP-10 on the integrity of corneal epithelial BM and integrin alpha3 beta1 and on wound healing in organ-cultured human corneas. Specific Aim 2. To identify by gene array analysis growth factors and cytokines abnormally expressed in diabetic and DR corneas and examine their effects on MMP-10 and wound healing in normal organ-cultured corneas. Specific Aim 3. To assess by gene array analysis the expression levels of various proteinases in diabetic, DR and normal human corneas. Identify and analyze additional proteinases with elevated expression in diabetic corneas. Specific Aim 4. To attempt blocking BM and integrin degradation in human diabetic and DR organ-culture corneas. Neutralizing antibodies to specific growth factors and proteinases (primarily, MMP-10), and various MMP inhibitors including clinically approved tetracyclines will be tested in organ-cultured corneas. These studies could lead to the development of novel therapeutics that would block the progression of diabetic keratopathy.