Abnormalities in Keratoconus Corneas

描述（由申请人提供）：圆锥角膜是一种角膜疾病 以基质过度变薄、严重的不规则散光为特征 和视力下降。它是角膜炎的主要适应症， 在美国境内移植。其发病机制的特点是， 降解酶的活性增加， 应激相关分子，增加局灶性纤维化和凋亡。的 引发这些变化或将它们联系在一起的潜在缺陷是 还不清楚。 在过去的三年里，我们应用了差分显示技术， 圆锥角膜cDNA智能合成及核酸芯片分析 细胞培养大约有3,000个基因被筛选出来， 正常人和圆锥角膜之间的表达。我们在两个病人身上发现了 不同的信号转导途径，（1）受体样蛋白酪氨酸 磷酸酶（白细胞共同抗原相关蛋白- LAR）和（2）a受体 酪氨酸激酶（ErbB 3），其配体（调蛋白）和下游因子，PYK 2， EPB-l和TOB。我们的数据还表明，KC角膜具有增加的诱导性， 一氧化氮合酶（iNOS）和过氧亚硝酸盐的积累， 一氧化氮的副产品我们假设KC角膜有缺陷， 它们处理自由基并上调这些信号的能力 转导途径。这导致异常的蛋白质磷酸化 模式，这在很大程度上有助于圆锥角膜的发病机制。测试 根据这一假设，我们提出以下具体目标： 具体目标#1将确定正常和非正常组织中的LAR亚型， 圆锥角膜和酪氨酸磷酸化模式特征性变化 由于活动。 特定目标#2将鉴定特定的ErbB家族成员和heregulin 同种型存在于圆锥角膜和体外KC细胞培养物中。的 核酸阵列数据显示PYK 2、JNK、EPB-1和TOB的上调 将在RNA（RT-PCR、北方分析、原位杂交）中进行确认 和蛋白质水平（免疫组织化学和Western印迹分析）。 具体目标#3将解决一氧化氮供体或过氧亚硝酸盐是否可以 影响酪氨酸磷酸酶（LAR）或酪氨酸激酶（ErbBIPYK 2/JNK） 途径。蛋白质由于硝化作用而发生变化的蛋白质 将识别过氧亚硝酸盐积累。 具体目标#4将确定是否加入调蛋白，一氧化氮 供体或过氧亚硝酸盐能够使正常细胞改变为 与KC相关的表型。 这些研究将为KC发病机制提供基本的见解， 为治疗提供依据。

DESCRIPTION (provided by applicant): Keratoconus is a corneal disorder characterized by excessive thinning of the stroma, severe irregular astigmatism and decreased visual acuity. It is a leading indication for corneal transplantation within the United States. Its pathogenesis is characterized by increased activities of degradative enzymes, altered processing of oxidative stress-related molecules, increased focal fibrosis and apoptosis. The underlying defect(s) that initiates these changes or ties them together is still not clear. During the past three years we have applied differential display technology, Smart cDNA synthesis and nucleic acid array analysis to keratoconus corneas and cell cultures. Approximately 3,000 genes have been screened for differential expression between normal and keratoconus. We found abnormalities in two distinct signal transduction pathways, (1) a receptor-like protein tyrosine phosphatase (leukocyte common antigen related protein- LAR) and (2) a receptor tyrosine kinase (ErbB3), its ligand (heregulin) and downstream factors, PYK2, EPB-l and TOB. Our data also show that KC corneas have increased inducible nitric oxide synthase (iNOS) and accumulation of peroxynitrite, a cytotoxic by-product of nitric oxide. We hypothesize that KC corneas have a defect in their ability to process free radicals and have up-regulation of these signal transduction pathways. This results in abnormal protein phosphorylation patterns, which contributes heavily to the pathogenesis of keratoconus. To test this hypothesis we propose the following specific aims: Specific Aim #1 will identify the LAR isoforms within the normal and keratoconus corneas and charactize changes in tyrosine phosphorylation patterns as a result of LAR activity. Specific Aim #2 will identify specific ErbB family members and heregulin isoforms present within keratoconus corneas and in vitro KC cell cultures. The nucleic acid array data demonstrating up-regulation of PYK2, JNK, EPB-l and TOB will be confirmed at the RNA (RT-PCR, Northern analyses, in situ hybridization) and protein levels (immunohistochemistry and Western blot analysis). Specific Aim #3 will address whether nitric oxide donors or peroxynitrites can affect the tyrosine phosphatase (LAR) or the tyrosine kinase (ErbBIPYK2/JNK) pathways. Proteins that undergo changes in nitration as a result of peroxynitrite accumulation will be identified. Specific Aim #4 will determine if the addition of heregulins, nitric oxide donors or peroxynitrites are capable of causing normal cells to change to the phenotype associated with KC. These studies will provide fundamental insights into KC pathogensis and may provide a basis for therapy.