Abnormalities in Keratoconus Corneas

描述(申请人提供)：圆锥角膜是一种角膜疾病 以基质过度变薄、严重不规则散光为特征 视力下降。这是角膜的主要适应症。 在美国境内进行移植。其发病机制的特点是 降解酶活性增加，氧化过程改变 应激相关分子，增加局灶性纤维化和细胞凋亡。这个 引发这些变化或将它们联系在一起的潜在缺陷(S)是 仍然不清楚。 在过去的三年里，我们应用了差异显示技术， 圆锥角膜和角膜SMART基因的合成及核酸阵列分析 细胞培养。已经对大约3000个基因进行了差异筛选 在正常角膜和圆锥角膜中的表达。我们发现其中两个有异常 不同的信号转导通路，(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(ErbB3)及其配体(Hereglin)及其下游因子PYK2， EPB-L和TOB。我们的数据还显示，KC角膜具有更高的诱导性 一氧化氮合酶(INOS)与过氧亚硝酸盐的蓄积 一氧化氮的副产物。我们假设KC角膜有缺陷 他们处理自由基并上调这些信号的能力 转导通路。这会导致蛋白质异常磷酸化。 模式，这在圆锥角膜的发病机制中起着重要作用。为了测试 在这一假设中，我们提出了以下具体目标： 具体目标#1将确定正常和 圆锥角膜及其特征性酪氨酸磷酸化模式的变化 作为LAR活动的结果。 具体目标#2将确定特定的ErbB家族成员和Heregine 异构体存在于圆锥角膜和体外培养的KC细胞中。这个 核酸阵列数据显示PYK2、JNK、EPB-L和TOB上调 将在RNA(RT-PCR、Northern分析、原位杂交)中得到确认 和蛋白水平(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(ErbBIPYK2/JNK) 小路。在硝化过程中发生变化的蛋白质 过氧亚硝酸盐的积累将被确定。 具体目标#4将确定是否添加Heregulins，一氧化氮 供体或过氧亚硝酸盐能够使正常细胞转变为 与KC相关的表型。 这些研究将为KC的发病机制提供基本的见解，并可能 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.