Mechanisms of Epithelial Alterations in Diabetic Cornea

描述（由申请人提供）：糖尿病视网膜病变一直是主要的 自1974年以来，美国的失明原因。这是显而易见的， 糖尿病眼微血管系统的进行性变化，导致 玻璃体内水肿、视网膜水肿、新生血管形成和视网膜脱离。 沿着视网膜、角膜、透镜和虹膜也受到糖尿病的影响。 许多糖尿病患者患有糖尿病性角膜病变， 糜烂、上皮脆性、异常伤口愈合和增加 易受伤。上皮-基质相互作用改变， 上皮基底膜（BM）缺陷可能导致糖尿病 角膜病变尽管糖尿病性角膜疾病的临床重要性， 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成分的表达和 已经在正常和糖尿病人角膜中分析了蛋白酶。我们表明 1.糖尿病视网膜病变（DR）角膜上皮细胞严重减少， 层粘连蛋白-1、层粘连蛋白-10、巢蛋白-1/巢蛋白以及层粘连蛋白-10/巢蛋白的BM免疫染色。 上皮整联蛋白α 3 β 1; 2. BM蛋白的基因表达和 整合素α 3 β 1在糖尿病和DR角膜上皮中没有改变; 3. 基质金属蛋白酶（MMP）-10的基因和蛋白表达增加， 糖尿病和DR角膜上皮和基质，MMP-3表达增加 在糖尿病和DR角膜基质中。数据显示， 角膜上皮BM在糖尿病中改变，尤其是DR， 特异性蛋白酶的活性，例如，MMP-10在细胞中表达， 上皮 我们的假设是糖尿病和DR患者的角膜上皮BM经历了 通过升高的蛋白酶，特别是MMP-10降解。蛋白酶表达 糖尿病患者体内激活的特异性生长因子可刺激其活性， 微环境这些改变可能构成了 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（MMP-10）对角膜基质完整性的影响。 上皮BM和整合素α 3 β 1以及对器官培养的伤口愈合的影响 人类角膜 具体目标2。通过基因芯片分析鉴定生长因子和细胞因子 在糖尿病和DR角膜中异常表达，并检查它们对 MMP-10与正常器官培养角膜的伤口愈合 具体目标3。通过基因阵列分析评估 糖尿病、DR和正常人角膜中的各种蛋白酶。识别和 分析在糖尿病角膜中表达升高的其他蛋白酶。 具体目标4。尝试阻断人BM和整合素降解 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MMP-10）以及各种MMP抑制剂 包括临床批准的四环素类将在器官培养的 角膜这些研究可能会导致新疗法的发展， 会阻止糖尿病性角膜病变的发展

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.